Selective Amplification of Nucleic Acid Sequences

ABSTRACT

The present invention relates to the field of nucleic acid sequence replication including PCR. Specifically, the present invention relates to methods and compositions for amplifying one or more target sequences from one or more template sequences. In particular, the present invention provides novel primer designs to enhance specificity of PCR reactions. The present invention also provides methods and compositions to perform the selection of specific sequence sections using specific primers and the amplification of all selected sequence sections using a pair of common primers in a single reaction tube.

PRIOR APPLICATIONS

This application claims priority to U.S. Ser. No. 61/963,211 filed Nov.26, 2013, which is hereby incorporated in its entirety into thisapplication.

FIELD OF INVENTION

The present invention relates to the field of nucleic acid sequencereplication including PCR. Specifically, the present invention relatesto methods and compositions for amplifying one or more target sequencesfrom one or more template sequences. In particular, the presentinvention provides novel primer designs to enhance specificity of PCRreactions. The present invention also provides methods and compositionsto perform the selection of specific sequence sections using specificprimers and the amplification of all selected sequence sections using apair of common primers in a single reaction tube.

BACKGROUND

Many biological and biomedical applications, such as genetic diseasescreening, personal medicine, forensic tests, and targeted sequencing,require replication and/or amplification of one or more selected nucleicacid sequence sections from a large pool of nucleic acid sequences, suchas genomic DNA from various living cells, nucleic acid sequence mixturesfrom metagenomic samples, and microbiome DNA from intestinal flora. PCRis a powerful tool to selectively amplify the nucleic acid sequences ofinterest in a nucleic acid mixture. For applications involving multiplesequences of interest multiplex PCR has been widely applied.Multiplexing PCR was first reported in 1988 by Chamberlain et al in“Deletion screening of the Duchenne muscular dystrophy locus viamultiplex DNA amplification” (1988) Nucleic Acids Res. 16: 11141-11156.However, conventional multiplex PCR is generally considered difficult toperform on more than a few dozen targets per reaction due to therequirement of significant optimization efforts that are described byHenegariu et al. (1997) “Multiplex PCR: Critical Parameters andStep-by-Step Protocol” BioTechniques 23: 504-511. The main challengeswith multiplex PCR include the formation of primer-dimers, non-uniformamplification of targets, and high rates of mispriming events, asdescribed by Edwards et al. (1994) “Multiplex PCR: advantages,development, and applications” Genome Res. 3:S65-75.

For the purpose of unifying primer-dependent PCR conditions, a variationof multiplex PCR has been described by A. P. Shuber et al. (1995) “ASimplified Procedure for Developing Multiplex PCRs” Genome Res. 5:488-493. Chimeric primers each containing a 3′ region complementary tosequence-specific recognition sites and a 5′ region made up of anunrelated 20-nucleotide sequence are used in multiplex PCR. Identicalreaction conditions, cycling times, and annealing temperatures weredemonstrated for any PCR primer pair comprising the chimeric motif. Thismethod was said to have helped eliminating the multiple optimizationsteps involved in developing multiplex PCR. However, the adjustment ofindividual primer concentrations was still required. The presentinvention provides a solution to eliminate the requirement for theindividual primer concentration adjustment.

For the purposes of reducing primer-dimer formation and reducingmispriming events, variations of multiplex PCR have been reported. Z.Lin et al. (1996), in “Multiplex genotype determination at a largenumber of gene loci” Proc. Natl. Acad. Sci. 93: 2582-2587, described amethod of converting multiplex amplification into uniplex amplificationso as to reduce primer-primer interaction. The process to implement themethod consists of three separate PCR rounds. In the first two PCRrounds locus specific primers containing 5′ universal tails or tags areused to attach the universal tails to the target sequences. Then, inround 3, all the universal tail tagged targets (26 genetic loci) areamplified simultaneously using one pair of universal primers that aresequence-matched to the universal tails. Round 1 and round 2 are similarto conventional multiplex PCR runs and a time-consuming optimizationprocess involving primer concentration adjustments is required.Additionally, the workflow consists of multiple hands-on steps includingthree separate PCR rounds and PCR product purifications between the PCRrounds. As comparison, the present invention provides a significantlysimplified workflow.

A DNA typing method, Minisatellite Variant Repeat mapping by PCR(MVR-PCR), is described by A. J. Jeffreys et al. (1991) “Minisatelliterepeat coding as a digital approach to DNA typing” Nature 354:204-209.Each PCR reaction includes three primers: a 5′ tagged variant repeatspecific primer at a low concentration of 10 to 20 nM, a minisatelliteflanking primer at a high concentration of 1 μM, and a tag primer thathas a sequence matching to the tagged section of the variant repeatspecific primer at a high concentration of 1 μM. The flanking primer hasa sequence matches to a flanking section of the minisatellite region.Recombinant Taq polymerase, AmpliTaq (Perkin-Elmer-Centus) is used. PCRreactions are cycled for 1.3 min denaturation at 96° C., 1 min annealingat 68° C., and 5 min extension at 70° C. for 18 cycles, followed by achase for 1 min at 67° C., and 10 min at 70° C. for 2 cycles on a DNAthermal cycler. In one reaction run more than 50 amplicons of differentlengths were produced each representing the distance between a specificvariant repeating unit and the flanking site. The production of theamplicons starts in a thermo cycle with the annealing of the variantrepeat specific primers to matching variant repeat units of theminisatellite region of a sample DNA. The annealed specific primers arethen extended beyond the flanking site forming the first extensionproducts by polymerase extension reaction. In the next thermo cycle, theflanking primers are annealed to the first extension products and areextended creating the second extension products with ends complementaryto tag sequence. From the next thermo cycle on the high concentrationflank primers and the tag primers work as a pair on the second extensionproducts to efficiently generate PCR products. Occasional internalpriming off the PCR products by the specific primers generates authenticshorter PCR products. This is the first work to demonstrate thefeasibility of tag-driven PCR by incorporating the tag primer at ahigher concentration relative to the tagged specific primer. On theother hand, the described approach is designed to reveal the patterns ofrepeating sequence units that share one target specific primer. Togetheronly three primers were used in each PCR reaction mixture. The work doesnot provide any obvious solution on how to handle a general type ofmultiplex PCR in which multiple target specific primers are involved andprimer-dimer formation must be minimized.

J. Brownie et al. (1997), in “the elimination of primer-dimeraccumulation in PCR” Nucleic Acids Res. 25: 3235-3241, described aHomo-Tag Assisted Non-Dimer System (HANDS) to reduce the formationprimer-dimers. Multiple tagged genome-specific primers at lowconcentrations and a single Tag or common primer at a high concentrationare used. Similar to Jeffreys method above, the Tag primer has the samesequence as that of the 5′ tail portion of the tagged genome-specificprimers. The authors suggested designing the genome-specific primerssuch that the Tm (melting temperature) of the Tag annealed to itscomplementary sequence is higher than that of the genome-specific primedduplex. This design enables a switch from genomic priming by thegenome-specific portions of the genome-specific primers at a lowannealing temperature to tail priming by the Tag at an elevatedannealing temperature. After two cycles of genomic priming, thecomplement of Tag sequence is incorporated into the amplicon ends. Thenthe annealing temperature is raised and subsequent amplifications arelargely driven by the Tag primers. All the amplicons produced have thesame pair of complementary ends. When the amplicons are short (100 to120 nucleotides), as with primer-dimers, the complementary ends tend togives rise to hairpin structures. The formation of these hairpinstructures outcompetes the annealing of further tag primers therebypreventing the accumulation of non-specific primer-dimer products.

A drawback of HANDS method is the product yield reduction of shorton-target sequences. By design the method inhibits the amplification ofshort sequences no matter if they are unwanted primer dimers or wantedon-target sequences. Indeed the reported data reveals a pattern ofsignificantly reduced product yields as the lengths of on-targetproducts decreased from 550 to 300 nucleotides. This makes the methodinadequate for applications involving on-target amplicon length below300 nucleotides. Such applications include sequence enrichment forhighly parallel sequencing uses.

Taq polymerase was used in both methods described by Jeffreys andBrownie shown above. Taq polymerase has a well-known 5′-3′ endonucleaseactivity (P. M. Holland et al. (1991) “Detection of specific polymerasechain reaction product by utilizing the 5′-3′ exonuclease activity ofThermus aquaticus DNA polymerase” Proc. Natl. Acad. Sci. 88 7276-7280)and 5′ flap endonuclease activity (V. Lyamichev et al. (1993)“Structure-specific endonucleolytic cleavage of nucleic acids byeubacterial DNA polymerases” Science 260:778-783). These nucleaseactivities cause degradation of double-stranded DNA that the polymeraseencounters while extending a DNA fragment. This imposes limitations onapplications where two or more target regions are in tandem or inpositional proximity. Primers hybridized to the middle of the tandemregions have high probability of being degraded and failing to producedesired amplicons. Such phenomenon is indeed observed in the datapresented in the above Jeffreys' publication. One aspect of the presentinvention is to overcome this limitation by utilizing newly availablepolymerases that lack 5′-3′ exonuclease activity and strand displacementactivity.

Another tag-driven PCR method for multiplex amplification applicationsis disclosed in B. Frey et al. (2013) “Methods and amplification oftarget nucleic acids using a multi-primer approach” US PatentApplication Publication US 2013/00045894 A1. Similar to the methods ofJeffreys and Brownie, two sets of primers including tagged targetspecific primers and common primers are used to amplify target nucleicacids in a single amplification reaction. A distinct reaction conditionfeature is that specific primer set concentration is suggested to be thesame or higher than that of common primer set. Consequently, reactionproduct contains shorter sequences flanked by the tagged specificprimers and longer sequences flanked by the common primers at comparableconcentration levels. The authors suggest separating the longersequences (the desired product) from the shorter sequences (undesiredproduct) before being used for corresponding applications. The presentinvention identified significantly different reaction conditions thatresult in clean products overwhelmingly dominated by the desired fulllength sequences.

The advances of high multiplex nucleic acid detection technologies suchas microarray and massively parallel sequencing have made it possible toanalyze hundreds, thousands, and up to millions of nucleic sequences inone test run. Successful applications of these detection technologiesoften share a common feature in that there is a requirement for anamplification of the regions of interests prior to actual detections.High multiplex amplification using surface immobilized primer pairs hasbeen described in various publications including A. Pemov et al. (2005)“DNA analysis with multiplex microarray-enhanced PCR”, Nucleic AcidsRes. 33: e11; L. S. Meuzelaar et al. (2007) “MegaPlex PCR: a strategyfor multiplex amplification”, Nat. Methods 4: 835-837; and referencesquoted therein. Multiple target specific primer pairs are immobilizedeither to a solid surface or inside a gel matrix through their 5′ ends.The immobilization is said to help avoid primer-dimer formation due tophysical separation of the primers. Each primer consists of a targetspecific priming section on 3′ side and a common priming section at 5′side. The immobilized primers are used to selectively replicate targetsequences and to incorporate common priming sections into the replicatedsequences in a solid-phase PCR process. Then a pair of common primers isused to amplify the solid-phase PCR products. The use of a single commonprimer pair eliminates target specific primer related amplificationbiases. However, the use of solid phase PCR complicates processworkflows. A simpler method is desirable.

Solution phase multiplex PCR using cleavable primers have been describedin K. E. Varley et al. (2008) “Nested Patch PCR enables highlymultiplexed mutation discovery in candidate Genes” Genome Res.18:1844-1850. The method relies on two rounds of target-specificenrichment. First, primer pairs are designed against each target, andthe mixture of primers used for a predetermined number of cycles ofmultiplex PCR. The primers contain uracil bases in place of thymine,such that post-amplification exposure to uracil DNA glycosylase,endonuclease VIII, and Exonuclease I effectively removes the primerregions from amplicons. For the second round of selection, Nested Patchadaptors are used. These adaptors consist of a double-stranded universalsegment and a single-stranded overhang that is target specific.Hybridization and ligation of Nested Patch adaptors to primer-depletedamplicons is followed by multi-template PCR amplification with primerscorresponding to the universal sequences. Because this ligation isdependent on sequences immediately internal to the original primers usedin the limited multiplex PCR, the Nested Patch adaptors conferadditional specificity. A variation of the method is describe by J.Leamon et al. (2012) “Methods and compositions for multiplex PCR” USPatent Application Publication US 2012/0295819 A1. The method omits thesecond round of selection by attaching common adapters using blunt endligation. These two versions of the method share a similar workflowrequiring multiple hands-on steps. Additionally, amplifications ofindividual targets in the first round multiplex PCR are carried out byindividual target specific primers. Inevitably primer related yielddifferences are exponentially amplified as the number of thermo cyclesincreases. This leads to non-uniform amplification of targets. Oneaspect of the present invention is to provide a simple workflow and toachieve uniform high multiplex amplification in solution phase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematic diagrams of three exemplary designs of thedisclosed omega primer. A. omega primer with internal loops; B. omegaprimer with hairpin loop; and C. omega primer with bulge loop.

FIG. 2 shows schematic diagrams of an exemplary design of the disclosedomega probe.

FIG. 3 schematically outlines an exemplary embodiment of relay PCRmethod using regular specific primers.

FIG. 4A schematically outlines an exemplary embodiment of relay PCRmethod using internal loop omega specific primers.

FIG. 4B schematically outlines an exemplary embodiment of relay PCRmethod using bulge loop omega specific primers.

FIG. 5 schematically outlines an exemplary embodiment of relay PCRmethod using multi-loop omega specific primers.

FIG. 6 schematically outlines an exemplary embodiment of singular primerextension relay PCR method using singular omega specific primers intarget selection.

FIG. 7 illustrates an exemplary embodiment of purification method ofthis invention.

FIG. 8 illustrates an exemplary embodiment of target immobilizationmethod of this invention.

FIG. 9 illustrates an exemplary embodiment of producing specific primersinvolving precursor amplification and activation.

FIG. 10 illustrates an exemplary embodiment of producing specificprimers involving PCR amplification and cap removal by dU digestion.

FIG. 11 illustrates an exemplary embodiment of producing specificprimers involving PCR amplification and cap removal by restrictiondigestion.

FIG. 12 shows a computation flow chart for primer design.

FIG. 13 illustrates an assembly of variant alleles in a genomic region.

FIG. 14 illustrates simultaneous equilibrium reactions of intramolecularfolding and intermolecular hybridization.

FIG. 15 illustrates simultaneous equilibrium reactions of intra-strandfolding and inter-strand hybridization involved in binding between anomega primer and a template.

FIG. 16 shows agarose gel electrophoresis images of example regular PCRand relay PCR products on a Lambda DNA sample.

Lane 1: Regular PCR, specific primer 1&2=500 nM, lambda DNA=10 fM

Lane 2: Relay PCR, specific primer 1&2=5 nM, common primer 1&2=500 nM,lambda DNA=10 fM

Lane 3: Relay PCR, specific primer 1&2=0.5 nM, common primer 1&2=500 nM,lambda DNA=10 fM

Lane L: is a DNA ladder run showing the sizes (in base pair or bp) ofcorresponding markers.

FIG. 17 shows agarose gel electrophoresis image of example relay PCRusing omega primers on a human genomic DNA sample. Lane 1 through lane 6shows the products of six individual PCR runs each involving a pair ofomega primers of a unique target region as shown in corresponding tablein Experiment II. The same common primer pairs were used in all six PCRruns. Lane 7 shows the result of no-specific primer control run. Lane Lis a DNA ladder showing the sizes (in base pair or bp) of correspondingmarkers.

FIG. 18 shows the experimental results of a multiplex relay PCR usingomega primers. A. shows agarose gel electrophoresis image of an examplemultiplex relay PCR using omega primers on a human genomic DNA sample.Lane 1 shows the product of a multiplex PCR run, which include 6 pairsof omega primers and one pair common primers that are used in theindividual relay PCR runs of FIG. 5. Lane L is a DNA ladder showing thesizes (in base pair or bp) of corresponding markers. B. shows a scatterplot of the sequencing read number distribution of the 6 expectedamplicons.

FIG. 19 shows high-throughput sequencing measurement results of ampliconread number distributions in multiplex PCR products. Figures A, B, and Care obtained by using specific omega primer concentrations of 1 nM, 0.2nM, and 0.04 nM per primer, respectively.

FIG. 20 shows the experimental results of multiplex relay PCR usingomega primers derived from microarray synthesized oligonucleotides. A.shows an agarose gel electrophoresis image of a PCR product ofmicroarray synthesized primer precursor templates. Lane 1 is the PCRproduct of a mixed template pool 204 oligonucleotide sequences. Lane Lis a DNA ladder showing the sizes (in base pair or bp) of correspondingmarkers. B. shows an agarose gel electrophoresis image of a multiplexrelay PCR product using the microarray derived primer precursor mixtureof FIG. 20A on a human genomic DNA sample. Lane 1 shows the product ofthe multiplex PCR run. Lane L is a DNA ladder showing the sizes (in basepair or bp) of corresponding markers.

FIG. 21 shows the experimental results of enzymatic preparation ofspecific primers and the use of the specific primers in multiplex relayPCR. A. shows an agarose gel electrophoresis image of PCR products ofspecific primer templates. Lane 1 is the PCR products of mixedtemplates. Lane L is a DNA ladder showing the sizes (in base pair or bp)of corresponding markers. B. lane 2 shows the gel image of restrictionenzyme digestion products. Lane 1 shows the original PCR product beforerestriction enzyme digestion. Lane L is a DNA ladder showing the sizes(in base pair or bp) of corresponding markers. C. shows the images ofrelay PCR products. Lane 1 shows the result of the positive controlderived from chemically synthesized specific primers. Lane 2 is therelay PCR products derived from enzymatically prepared specific primers.Lane 3 is the result of negative control. Lane L is a DNA ladder showingthe sizes (in base pair or bp) of corresponding markers.

DEFINITIONS

Term “tube” refers to a vessel in which PCR or any other types ofbimolecular reactions take place. The “tube” may be made of plastic andin form of micro tubes or Eppendorf tubes. The “tube” may also be madeof glass, silicone, silicon, and metals and be a part of microfabricateddevices.

Terms “target”, “target sequence”, and their derivatives refer to anysingle or double-stranded nucleic acid sequence that is suspected orexpected to be present in a sample and is designated to be selected,analyzed, examined, probed, captured, replicated, synthesized, and/oramplified using any appropriate methods.

Term “library”, “DNA library” and their derivatives, as used herein,refer to a collection of DNA fragments or DNA sequences that is subjectto parallel sample preparation and/or parallel detection assayprocesses. In some embodiments, such as multiplex amplification, targetsequences are selectively amplified using target specific primers andform a target sequence library.

Term “sample” refers to any specimen, culture and the like that issuspected of including a target. The sample can include any biological,clinical, surgical, agricultural, atmospheric or aquatic-based specimencontaining one or more nucleic acids. The sample can include any type ofspecimens such as cheek tissue, whole blood, dried blood spot, organtissue, plasma, urine, feces, skin, and hair. The term also includes anyisolated nucleic acid sample such as genomic DNA from fresh-frozen orformalin-fixed paraffin-embedded tissues.

Terms “synthesize”, “synthesizing”, and their derivatives refergenerally to a reaction involving nucleotide polymerization by apolymerase, optionally in a template-dependent fashion. Polymerasessynthesize an oligonucleotide via transfer of a nucleoside monophosphatefrom a nucleoside triphosphate (NTP), deoxynucleoside triphosphate(dNTP), or dideoxynucleoside triphosphate (ddNTP) to the 3′ hydroxyl ofan extending oligonucleotide chain. For the purposes of this disclosure,synthesizing includes the serial extension of a hybridized primer viatransfer of a nucleoside monophosphate from a deoxynucleosidetriphosphate.

Terms “oligo”, “oligonucleotide”, and their derivatives refer to short,single-stranded nucleic acid sequences including DNA, RNA, DNA-RNAhybrid, and various modification group containing molecules. They can beproduced or synthesized by chemical methods, enzymatic methods, orcombinations of chemical and enzymatic methods.

Term “extension” and its variants, as used herein, when used inreference to a given primer, comprise any in vivo or in vitro enzymaticactivity characteristic of a given polymerase that relates topolymerization of one or more nucleotides onto an end of an existingnucleic acid molecule. Typically but not necessarily such primerextension occurs in a template-dependent fashion; duringtemplate-dependent extension, the order and selection of bases is drivenby established base pairing rules, which can include Watson-Crick typebase pairing rules or alternatively (and especially in the case ofextension reactions involving nucleotide analogs) by some other type ofbase pairing paradigm. In one non-limiting example, extension occurs viapolymerization of nucleotides on the 3′OH end of the nucleic acidmolecule by the polymerase.

Terms “amplify”, “amplifying”, “amplification”, and their derivativesrefer generally to any action or process whereby at least a portion of anucleic acid molecule (referred to as a template nucleic acid molecule)is replicated or copied into at least one additional nucleic acidmolecule. The additional nucleic acid molecule optionally includessequence that is substantially identical or substantially complementaryto at least some portion of the template nucleic acid molecule. Thetemplate nucleic acid molecule can be single-stranded or double-strandedand the additional nucleic acid molecule can independently besingle-stranded or double-stranded. In some embodiments, amplificationincludes a template-dependent in vitro enzyme-catalyzed reaction for theproduction of at least one copy of at least some portion of the nucleicacid molecule or the production of at least one copy of a nucleic acidsequence that is complementary to at least some portion of the nucleicacid molecule. Amplification optionally includes linear or exponentialreplication of a nucleic acid molecule. In some embodiments, suchamplification is performed using isothermal conditions; in otherembodiments, such amplification can include thermocycling. In someembodiments, the amplification is a multiplex amplification thatincludes the simultaneous amplification of a plurality of targetsequences in a single amplification reaction. At least some of thetarget sequences can be situated on the same nucleic acid molecule or ondifferent target nucleic acid molecules included in the singleamplification reaction. In some embodiments, “amplification” includesamplification of at least some portion of DNA- and RNA-base nucleicacids alone, or in combination. The amplification reaction can includesingle or double-stranded nucleic acid substrates and can furtherincluding any of the amplification processes known to one of ordinaryskill in the art. In some embodiments, the amplification reactionincludes polymerase chain reaction (PCR).

Term “primer” and its derivatives refer to any polynucleotide oroligonucleotide that can hybridize to a target sequence of interest. Insome embodiments of this disclosure, at least 3′ end portion of a primeris complementary to a portion of the target sequence. Typically, theprimer acts as a point of initiation for amplification or synthesis whenexposed to amplification or synthesis conditions; such amplification orsynthesis can occur in a template-dependent fashion and optionallyresults in formation of a primer extension product that is complementaryto at least a portion of the target sequence. Exemplary amplification orsynthesis conditions can include contacting the primer with apolynucleotide template (e.g., a template including a target sequence),nucleotides and an inducing agent such as a polymerase at a suitabletemperature, salt concentration, and pH to induce polymerization ofnucleotides onto an end of the target-specific primer. A primer can bepaired with a compatible primer within an amplification or synthesisreaction to form a primer pair consisting of a forward primer and areverse primer. In some embodiments, the forward primer includes a 3′portion substantially complementary to at least a portion of a strand ofa nucleic acid molecule and the reverse primer includes a 3′ portionsubstantially identical to at least of portion of the strand. In someembodiments, the forward primer and the reverse primer are capable ofhybridizing to opposite strands of a nucleic acid duplex. Optionally,the forward primer primes synthesis of a first nucleic acid strand, andthe reverse primer primes synthesis of a second nucleic acid strand,wherein the first and second strands are substantially complementary toeach other, or can hybridize to form a double-stranded nucleic acidmolecule.

Term “specific primer” and “target-specific primer” refer to a singlestranded oligonucleotide that includes a 3′ specific section that issubstantially complementary or substantially identical to at least aportion of a nucleic acid molecule that includes a target sequence. Insome embodiments, the specific primer includes two or more specificsections that are substantially complementary or substantially identicalto portions of a nucleic acid molecule that includes a target sequence.In some embodiments, the specific primer includes at least a commonsegment. The common segment is a sequence segment that is designed to beshared in plurality of primers. In some embodiments, the common segmentis located between two specific sections. In some embodiments, thecommon segment is located at 5′ end portion of the specific primer.

Term “common primer” and “library primer” refer to a single strandedoligonucleotide that includes a 3′ section that is substantiallycomplementary or substantially identical to at least a portion of acommon segment of a nucleic acid molecule. In some embodiments, thecommon segment of the nucleic acid molecule is in a PCR product of atarget sequence. In some embodiments, the common segment of the nucleicacid molecule is in a primer extension product of a target sequence.

Term “hybridization” is consistent with its use in the art, andgenerally refers to the process whereby two nucleic acid moleculesundergo base pairing interactions. Two nucleic acid molecules are saidto be hybridized when any portion of one nucleic acid molecule is basepared with any portion of the other nucleic acid molecule; it is notnecessarily required that the two nucleic acid molecules be hybridizedacross their entire respective lengths and in some embodiments, at leastone of the nucleic acid molecules can include portions that are nothybridized to the other nucleic acid molecule.

Term “polymerase chain reaction” or “PCR” refers to the method of K. B.Mullis U.S. Pat. Nos. 4,683,195 and 4,683,202, hereby incorporated byreferences, which describe a method for increasing the concentration ofa segment of a polynucleotide of interest in a mixture of genomic DNAwithout cloning of purification. This process of amplifying thepolynucleotide of interest consists of introducing a large excess of twooligonucleotide primers to the DNA mixture containing the desiredpolynucleotide of interest, followed by a precise sequence of thermalcycling in the presence of a DNA polymerase. The two primers arecomplementary to their respective strands of the double strandedpolynucleotide of interest. To effect amplification, the mixture isdenatured and the primers then annealed to their complementary sequenceswithin the polynucleotide of interest molecules. Following annealing,the primers are extended with a polymerase to form a new pair ofcomplementary strands. The steps of denaturation, primer annealing andpolymerase extension can be repeated many times (i.e., denaturation,annealing and extension constitute one “cycle”, there can be numerous“cycles”) to obtain a high concentration of an amplified segment of thedesired polynucleotide of interest. The length of the amplified segmentof the desired polynucleotide of interest (amplicon) is determined bythe relative positions of the primers with respect to each other, andtherefore, this length is a controllable parameter. By virtue ofrepeating the process, the method is referred to as the “polymerasechain reaction” (hereinafter “PCR”). Because the desired amplifiedsegments of the polynucleotide of interest become the predominantnucleic acid sequences (in terms of concentration) in the mixture, theyare said to be “PCR amplified”.

Term “single-strand PCR” and “linear amplification reaction” refer to anamplification reaction that uses only one primer per primer specificsequence set. The “primer specific sequence” refers to a nucleic acidsequence containing at least one primer complementary section. A primerspecific sequence set contains one or more primer specific sequences.

Term “polymerase” and its derivatives generally refer to any enzyme thatcan catalyze the polymerization of nucleotides into a nucleic acidstrand. Typically but not necessarily, such nucleotide polymerizationcan occur in a template-dependent fashion. Such polymerase can includewithout limitation naturally occurring polymerases and any subunits andtruncations thereof, mutant polymerases, variant polymerases,recombinant, fusion or otherwise engineered polymerases, chemicallymodified polymerases, synthetic molecules or assemblies, and anyanalogs, derivatives or fragments thereof that retain the ability tocatalyze such polymerization. Optionally, the polymerase can be a mutantpolymerase comprising one or more mutations involving the replacement ofone or more amino acids with other amino acids, the insertion ordeletion of one or more amino acids from the polymerases, or the linkageof parts of two or more polymerases. Some exemplary polymerases includewithout limitation DNA polymerases and RNA polymerases. The term“polymerase” and its variants, as used herein, also refers to fusionproteins comprising at least two portions linked to each other, wherethe first portion comprises a peptide that can catalyze thepolymerization of nucleotides into a nucleic acid strand and is linkedto a second portion that comprises a second polypeptide. In someembodiments, the second polypeptide can include a reporter enzyme or aprocessivity-enhancing domain. Optionally, the polymerase can possess 5′exonuclease activity or terminal transferase activity. In someembodiments, the polymerase can be optionally reactivated, for examplethrough the use of heat, chemicals or re-addition of new amounts ofpolymerase into a reaction mixture. In some embodiments, the polymerasecan include thermo stable, hot-start, high-fidelity, 3′ to 5′ nucleaseactivity, 5′ to 3′ nuclease activity, and strand displacement activity.

Term “multiplex amplification”, “multiplex PCR” and their derivativesrefer to selective and non-random amplification of two or more targetsequences within a sample using at least one target specific primer. Insome embodiments, multiplex amplification is performed such that some orall of the target sequences are amplified within a single reactionvessel. The “plexity” or “plex” of a given multiplex amplificationrefers generally to the number of different target-specific sequencesthat are amplified during that single multiplex amplification.

Term “GC content” refers to the cytosine and guanine content of anucleic acid molecule.

Term “DNA barcode” and its derivatives, refers generally to a uniqueshort (4-14 nucleotide) nucleic acid sequence within a common primerthat can act as a ‘key’ to distinguish or separate a plurality ofamplified target sequences in a sample.

Term “complementary” and “complement” and their variants refer to anytwo or more nucleic acid sequence (e.g., portions or entireties oftemplate nucleic acid molecules, target sequence and/or primers) thatcan undergo cumulative base pairing at two or more individualcorresponding positions in antiparallel orientation, as in a hybridizedduplex. Such base pairing can proceed according to any set ofestablished rules, for example according to Watson-Crick base pairingrules or according to some other base pairing paradigm. Optionally therecan be “complete” complementarity between a first and a second nucleicacid sequence where each nucleotide in the first nucleic acid sequencecan undergo a stabilizing base pairing interaction with a nucleotide inthe corresponding antiparallel position on the second nucleic acidsequence. “Partial” complementarity describes nucleic acid sequences inwhich at least 20%, but less than 100%, of the residues of one nucleicacid sequence are complementary to residues in the other nucleic acidsequence. “Partial” complementarity also describes nucleic acidsequences in which at least 20%, but less than 100%, of the residues ofa section of interest of one nucleic acid sequence are complementary toresidues in the other nucleic acid sequence.

Term “association fraction”, “binding coefficient”, “fraction ofbinding”, and their derivatives refer to the fraction of a templatebeing hybridized with corresponding primer, a target being hybridizedwith corresponding probe, or one section being hybridized with anothersection of the same nucleic acid sequence. The calculation of theassociation fraction is described by Miura (Miura el al. (2005) “A novelstrategy to design highly specific PCR primers based on the stabilityand uniqueness of 3′-end subsequences” Bioinformatics 21 4363-4370) andin specifications of this disclosure.

Term “parallel sequencing”, “massive parallel sequencing”,“high-throughput sequencing”, “next generation sequencing” and theirvariants refer to sequencing technologies that parallelize thesequencing process, producing thousands or millions of sequencesconcurrently. Various methodologies and processes involved in thetechnology are described by Mardis E R (2008) “Next-generation DNAsequencing methods”, Annu Rev Genomics Hum Genet 9: 387-402; and by M.L. Metzker (2010) “Sequencing technologies—the next generation”, NatureReview Genetics 11:31-46.

Term “priming region”, “priming section”, and their derivatives refer toa section of a target sequence of interest that is designed to besubstantially hybridized with a corresponding primer.

Term “probe sequence”, “probe”, and their derivatives refer to a nucleicacid sequence that is designed to be hybridized with a target sequenceof interest for the purposes of detection, capture, and enrichment. Insome embodiments, a probe contains one or more fluorescence dye groups.In some embodiments, the probe contains one or more fluorescencequencher groups. In some embodiments, the probe contains one or morefluorescence quencher groups. In some embodiments, the probe containsone or more donor and/or acceptor fluorophore groups. In someembodiments, the probe sequence is a free molecule in a solution. Insome embodiments, the probe sequence is immobilized on a bead surface.In some embodiments, the probe sequence is immobilized on asubstantially flat surface. In some embodiments, the probe sequence isembedded in gel.

Term “specificity” generally refers to the fraction of correctlyreported events among all reported events. In some embodiments, the termrefers to the fraction of the primers correctly extended on the intendedtargets among all primers under consideration that have extended inprimer extension reactions. In some embodiments, the term refers to thefraction of correct PCR products among all PCR products produced in PCRreactions. In some embodiments, the term refers to the fraction ofperfectly matched probe-target pairs among all probe-target pairs inhybridization reactions.

Term “variant”, “sequence variation”, and their derivatives refer to asequence section of a sample of interest that is different from thesequence section at the same location in a reference sample or in areference sequence. The variation includes point and structuralvariation. For the purpose of this disclosure, the variation can beeither disease related or non-disease related. The point variationincludes single-nucleotide polymorphism or SNP. The structural variationincludes short insertion, short deletion, large insertion, largedeletion, indel (a deletion followed by an insertion), substitution,duplications, inversion, translocations, or any other types ofvariations.

Term “captured region”, “capture region”, and their derivatives refer tothe region of a target sequence sandwiched between two paired primersincluding the target specific sections of the primers.

SUMMARY

It is the object of the present invention to provide a new and improvedPCR method that is simple to perform, has improved sequence specificity,has low primer related amplification bias, and is suitable for multiplexamplification uses.

One application aspect of the present invention relates to the field oftarget enrichment for massive parallel sequencing applications. Targetsequences of interest are selected from genomic samples, amplified, andflanked with sequencing priming sections containing optional DNAbarcodes. Another application aspect of the present invention relates tofield of target enrichment for DNA array genotyping applications. Targetsequences of interest are selected from genomic samples, amplified, andlabeled with fluorescence dyes or with conjugation ligands. Technicalapproaches in the target enrichment methods of the two applicationsshare many similarities. Extensive technology reviews of the field havebeen provided by E. H. Turner et al. (2009) “Methods for genomicpartitioning”, Annu. Rev. Genomics Hum. Genet. 10:263-284 and byMemanova et al. (2010) “Target-enrichment strategies for nextgenerationsequencing” Nat. Methods 7, 111-118. Prevailing methods includemultiplex PCR, capture-by-circularization, and capture-by-hybridization.The performance of a method is mainly evaluated by capture specificity,uniformity, multiplexity, input requirements, scalability, workflowsimplicity, and cost. The present invention provides methods andcompositions for target enrichment with significantly improvedperformances.

The present invention relates to oligonucleotide primers having a 3p armwhich includes a 3′ end and a 5′ end, a loop section and a 5p arm havinga 3′ end and a 5′ end, where the 5p arm hybridizes to a DNA template andwherein the 3p arm hybridizes to the DNA template and provides sequencespecificity for polymerase extension and where the loop section islocated between the 5p arm and the 3p arm and does not bind the DNAtemplate.

The present invention relates to stable hybridization structurescomprising a designed oligonucleotide and a target nucleic acid, whereinthe designed sequence and the target sequence form a stable, where thehybridization structure has one or more single stranded loops and two ormore duplex segments wherein each loop is located between the duplexsegments.

The present invention relates to methods of amplifying a target nucleicacid comprising providing a first specific primer, a second specificprimer, a first common primer, a second common primer a target nucleicacid, a polymerase and nucleotides, performing a target selectioncomprising two cycles of a first thermocycling routine comprising andenaturation step, annealing step and an extension step and performingamplification comprising two or more cycles of a second thermocyclingroutine comprising an denaturation step, annealing step and an extensionstep thereby amplifying the target nucleic acid.

The present invention relates to methods of amplifying two or moredifferent target nucleic acids comprising providing a first set ofspecific primers containing two specific primers each primerspecifically designed for a first target nucleic acid, a second set ofspecific primers, each primer specifically designed for a second targetnucleic acid, a first common primer, a second common primer and a targetnucleic acid; performing two cycles of a first thermocycling routinecomprising an denaturation step, annealing step and an extension step;and performing two or more cycles of a second thermocycling routinecomprising an denaturation step, annealing step and an extension stepthereby amplifying the target nucleic acid.

The present invention relates to methods of purifying PCR productscomprising adding to a mixture of PCR reaction components comprisestarget sequences, a first common primer and a second common primer, DNAfragments, polymerase, PCR buffer solution wherein the target sequencesare flanked with priming segments that are either identical orcomplementary to the first common primer and the second common primerand wherein the fragments do not contain priming segments and whereinthe second common primer comprises a priming segment, a modifier segmentand a tag segment probe grafted beads wherein the probe has a sequencethat is substantially complementary to that of tag segment andfacilitates the capture of the PCR product by the beads 721 throughhybridization.

The present invention relates to methods of sequence library preparationcomprising amplifying a target sequence with a first common primer and asecond common primer each common primer comprising a priming segment,modifier segment and tag segment to produce PCR products containing asingle-stranded tag applying a guide solution to the substrate therebyhybridizing the probes to the substrate to produce guide/probe pairs onthe substrate surface, washing away excess guide solution and adding thesingle-strand tag containing PCR products to the substrate therebyco-hybridizing the single-strand tag, a guide, and the probe on thesubstrate surface.

DETAILED DESCRIPTION Target Specific Primer

One aspect of the invention relates to a primer form, called omegaprimer. FIG. 1 shows schematic diagrams of three exemplary structuraldesigns of the omega primer 100, 110, and 120. Each primer comprisesthree functional sections including a 3p arm 101, 111, and 121, a loop102, 112, and 122, and a 5p arm 103, 113, and 123. One function of the3p arm 101, 111, and 121 is to hybridize to a DNA template 104 and toprovide a starting point for polymerase extension reaction. The 5p arm103, 113, and 123 stabilizes the binding between the omega primer andthe DNA template. The loop 102, 112, and 122 provides a separationbetween the two arms. FIG. 1A schematically illustrates an omega primer100 with internal loops 102 and 105. The internal loops aresingle-stranded nucleic acid sequence sections on both primer 100 andtemplate 104 strands between the double-stranded 3p arm 101 and 5p arm103 sections. FIG. 1B schematically illustrates an omega primer 110 witha hairpin loop. The hairpin loop is formed by the primer sequence,comprising a single-stranded loop 112 and a double-stranded stem 113.FIG. 1C schematically illustrates an omega primer 120 with a bulge loop122. This bulge loop structure is formed solely inside the primer. Thesestructures are formed by hybridization interactions between primer andtemplate sequences and can be designed with theoretical calculations bythose of skilled in the art (see J. SantaLucia Jr. et al. (2004) “Thethermodynamics of DNA structural motifs” Annu. Rev. Biophys. Biomol.Struct. 33:415-440).

The disclosed omega primers provide desirable features and/or propertiesin various applications. One aspect of the invention is the utilizationof two separate binding sections to balance priming specificity andbinding strength. In one exemplary application, the 5p arm is designedto have higher binding energy than 3p arm has so that 5p arm mayinitiate and sustain binding while 3p arm checks sequence specificityfor polymerase extension. A successful priming reaction requires thehybridizations of the two separate sections. This reduces the chance foroff-target priming and results in highly specific primer designs. Thebinding strength of the 5p arm compared to the binding strength of the3p arm may be 1.1 times greater, or 1.2 times greater or 1.3 timesgreater or 1.4 times greater or 1.5 times greater of 1.6 times greateror 1.7 times greater, or 1.8 times greater or 1.9 times greater or 2times greater or 2.5 times greater of 3 times greater or 5 times greateror 10 times greater. The binding strength of the 5p arm compared to thebinding strength of the 3p arm may be between 1.1 to 100 times greater,1.1 to 50 times greater, 1.1 to 25 times greater, 1.1 to 20 timesgreater, 1.1 to 15 times greater, 1.1 to 10 times greater, 1.1 to 5times greater, 1.5 to 100 times greater, 1.5 to 50 times greater, 1.5 to25 times greater, 1.5 to 20 times greater, 1.5 to 15 times greater, 1.5to 10 times greater, 1.5 to 5 times greater, 2.0 to 100 times greater,2.0 to 50 times greater, 2.0 to 25 times greater, 2.0 to 20 timesgreater, 2.0 to 15 times greater, 2.0 to 10 times greater, 2.0 to 5times greater, 5.0 to 100 times greater, 5.0 to 50 times greater, 5.0 to25 times greater, 5.0 to 20 times greater, 5.0 to 15 times greater, 5.0to 10 times greater, 5.0 to 5 times greater.

Another aspect of this design is the ability to make the omega primerstolerant to certain template sequence variations at designated primingsite. This feature is highly desirable in assays designed for coveringgeneral populations. For example, the 1000 Genomes Project reported avalidated haplotype map of 38 million single nucleotide polymorphism(SNP) variants in human populations (The 1000 Genomes Project Consortium(2012) “An integrated map of genetic variation from 1,092 human genomes”Nature 491:56-65). This means one SNP variant in an average of less than200 nucleotides in human genome of 6 billion diploid nucleotides long.The current version NCBI dbSNP for human contains 74 million SNPvariants with genotype, meaning one SNP variant in average of every 40nucleotides in human genome of 3 billion nucleotides long. SNP is themost abundant form of variant in human genome. These variants imposechallenges to PCR primer design since a primer designed for a referencetarget sequence may not work well when it is applied to an actual targetsample that happens to contain one or more variants in correspondingpriming region. Due to the high density of the variants in generalpopulations, it is desirable to have primers that are able to toleratethe variants so as to maximize the accessible regions for priming in thetarget sequence. One aspect of this disclosure is to include thevariants into consideration in primer design. In designing omega primersthe 5p arm length should be sufficiently long that the binding betweenthe 5p arm and all anticipated variant target alleles is stable at thecorresponding reaction temperature. The 3p arm length should be justlong enough to achieve a stable binding with the reference targetallele. Thus, the 5p arm serves as an anchor and the 3p arm checks forcorrectness of the priming site. This design strategy significantlyexpands the assessable regions for priming in the target sequence,results in variant tolerant primers, and yet produces high primingspecificity. The 5p arm may be at least 10 nucleotides long, or at least15 nucleotides long or at least 20 nucleotides long, or at least 25nucleotides, or at least 30 nucleotides long, or at least 35 nucleotideslong or at least 40 nucleotides long, or at least 45 nucleotides long,or at least 50 nucleotides long, or at least 55 nucleotides long or atleast 60 nucleotides long, or at least 65 nucleotides or at least 70nucleotides long, or at least 75 nucleotides long or at least 80nucleotides long, or at least 85 nucleotides, or at least 90 nucleotideslong, or at least 95 nucleotides long or at least 100 nucleotides long,or at least 125 nucleotides long, or at least 150 nucleotides long, orat least 200 nucleotides long or at least 250 nucleotides long. The 5parm may be between 10 to 200 nucleotides in length, or be between 10 to150 nucleotides in length, or be between 10 to 100 nucleotides inlength, or be between 10 to 90 nucleotides in length, or between 10 to80 nucleotides in length, or be between 10 to 70 nucleotides in length,or be between 10 to 60 nucleotides in length, or be between 10 to 50nucleotides in length, or between 10 to 40 nucleotides in length, or bebetween 10 to 30 nucleotides in length, or be between 10 to 20nucleotides in length, or be between 15 to 200 nucleotides in length, orbe between 15 to 150 nucleotides in length, or be between 15 to 100nucleotides in length, or be between 15 to 90 nucleotides in length, orbetween 15 to 80 nucleotides in length, or be between 15 to 70nucleotides in length, or be between 15 to 60 nucleotides in length, orbe between 15 to 50 nucleotides in length, or between 15 to 40nucleotides in length, or be between 15 to 30 nucleotides in length, orbe between 15 to 20 nucleotides in length, or between 20 to 200nucleotides in length, or be between 20 to 150 nucleotides in length, orbe between 20 to 100 nucleotides in length, or be between 20 to 90nucleotides in length, or between 20 to 80 nucleotides in length, or bebetween 20 to 70 nucleotides in length, or be between 20 to 60nucleotides in length, or be between 20 to 50 nucleotides in length, orbetween 20 to 40 nucleotides in length, or be between 20 to 30nucleotides in length, or between 25 to 200 nucleotides in length, or bebetween 25 to 150 nucleotides in length, or be between 25 to 100nucleotides in length, or be between 25 to 90 nucleotides in length, orbetween 25 to 80 nucleotides in length, or be between 25 to 70nucleotides in length, or be between 25 to 60 nucleotides in length, orbe between 25 to 50 nucleotides in length, or between 25 to 40nucleotides in length, or be between 25 to 30 nucleotides in length, orbetween 30 to 200 nucleotides in length, or be between 30 to 150nucleotides in length, or be between 30 to 100 nucleotides in length, orbe between 30 to 90 nucleotides in length, or between 30 to 80nucleotides in length, or be between 30 to 70 nucleotides in length, orbe between 30 to 60 nucleotides in length, or be between 30 to 50nucleotides in length, or between 30 to 40 nucleotides in length, or bebetween 35 to 200 nucleotides in length, or be between 35 to 150nucleotides in length, or be between 35 to 100 nucleotides in length, orbe between 35 to 90 nucleotides in length, or between 35 to 80nucleotides in length, or be between 35 to 70 nucleotides in length, orbe between 35 to 60 nucleotides in length, or be between 35 to 50nucleotides in length, or between 35 to 40 nucleotides in length, or bebetween 40 to 200 nucleotides in length, or be between 40 to 150nucleotides in length, or be between 40 to 100 nucleotides in length, orbe between 40 to 90 nucleotides in length, or between 40 to 80nucleotides in length, or be between 40 to 70 nucleotides in length, orbe between 40 to 60 nucleotides in length, or be between 40 to 50nucleotides in length.

The 3p arm may be at least 5 nucleotides long, or at least 6 nucleotideslong or at least 7 nucleotides long, or at least 8 nucleotides, or atleast 9 nucleotides long, or at least 10, or at least 11 nucleotideslong or at least 12 nucleotides long, or at least 13 nucleotides long,or at least 14 nucleotides long, or at least 15 nucleotides long or atleast 20 nucleotides long, or at least 25 nucleotides or at least 30nucleotides long, or at least 35 nucleotides long or at least 40nucleotides long, or at least 45 nucleotides, or at least 50 nucleotideslong, or at least 55 nucleotides long or at least 60 nucleotides long,or at least 65 nucleotides long, or at least 70 nucleotides long, or atleast 80 nucleotides long or at least 90 nucleotides long. The 3p armmay be between 5 to 100 nucleotides in length, or be between 5 to 90nucleotides in length, or between 5 to 80 nucleotides in length, or bebetween 5 to 70 nucleotides in length, or be between 5 to 60 nucleotidesin length, or be between 5 to 55 nucleotides in length, or between 5 to50 nucleotides in length, or be between 5 to 45 nucleotides in length,or be between 5 to 40 nucleotides in length, or be between 5 to 35nucleotides in length, or be between 5 to 30 nucleotides in length, orbe between 5 to 25 nucleotides in length, or be between 5 to 20nucleotides in length, or between 5 to 15 nucleotides in length, or bebetween 5 to 10 nucleotides in length, or be between 6 to 100nucleotides in length, or be between 6 to 90 nucleotides in length, orbetween 6 to 80 nucleotides in length, or be between 6 to 70 nucleotidesin length, or be between 6 to 60 nucleotides in length, or be between 6to 55 nucleotides in length, or between 6 to 50 nucleotides in length,or be between 6 to 45 nucleotides in length, or be between 6 to 40nucleotides in length, or be between 6 to 35 nucleotides in length, orbe between 6 to 30 nucleotides in length, or be between 6 to 25nucleotides in length, or be between 6 to 20 nucleotides in length, orbetween 6 to 15 nucleotides in length, or be between 6 to 10 nucleotidesin length, or between 7 to 100 nucleotides in length, or be between 7 to90 nucleotides in length, or between 7 to 80 nucleotides in length, orbe between 7 to 70 nucleotides in length, or be between 7 to 60nucleotides in length, or be between 7 to 55 nucleotides in length, orbetween 7 to 50 nucleotides in length, or be between 7 to 45 nucleotidesin length, or be between 7 to 40 nucleotides in length, or be between 7to 35 nucleotides in length, or be between 7 to 30 nucleotides inlength, or be between 7 to 25 nucleotides in length, or be between 7 to20 nucleotides in length, or between 7 to 15 nucleotides in length, orbe between 7 to 10 nucleotides in length, be between 10 to 100nucleotides in length, or be between 10 to 90 nucleotides in length, orbetween 10 to 80 nucleotides in length, or be between 10 to 70nucleotides in length, or be between 10 to 60 nucleotides in length, orbe between 10 to 55 nucleotides in length, or between 10 to 50nucleotides in length, or be between 10 to 45 nucleotides in length, orbe between 10 to 40 nucleotides in length, or be between 10 to 35nucleotides in length, or be between 10 to 30 nucleotides in length, orbe between 10 to 25 nucleotides in length, or be between 10 to 20nucleotides in length, or between 10 to 15 nucleotides in length, orbetween be between 15 to 100 nucleotides in length, or be between 15 to90 nucleotides in length, or between 15 to 80 nucleotides in length, orbe between 15 to 70 nucleotides in length, or be between 15 to 60nucleotides in length, or be between 15 to 55 nucleotides in length, orbetween 15 to 50 nucleotides in length, or be between 15 to 45nucleotides in length, or be between 15 to 40 nucleotides in length, orbe between 15 to 35 nucleotides in length, or be between 15 to 30nucleotides in length, or be between 15 to 25 nucleotides in length, orbe between 15 to 20 nucleotides in length.

There can be various embodiments of the disclosed omega primer designs.For example, a primer can consist of three or more binding sections thatare separated by two or more loops. It is desirable to distribute thebinding feature of a primer into two or more segmented binding sectionsseparated by loop sections for the purpose of improving primingspecificity, modulating binding strength, inserting or incorporatingspecific sequences, and/or obtaining other desirable functions. The loopmay be at least 5 nucleotides long, or at least 6 nucleotides long or atleast 7 nucleotides long, or at least 8 nucleotides, or at least 9nucleotides long, or at least 10, or at least 11 nucleotides long or atleast 12 nucleotides long, or at least 13 nucleotides long, or at least14 nucleotides long, or at least 15 nucleotides long or at least 20nucleotides long, or at least 25 nucleotides or at least 30 nucleotideslong, or at least 35 nucleotides long or at least 40 nucleotides long,or at least 45 nucleotides, or at least 50 nucleotides long, or at least55 nucleotides long or at least 60 nucleotides long, or at least 65nucleotides long, or at least 70 nucleotides long. The loop may bebetween 7 to 100 nucleotides in length, or be between 7 to 90nucleotides in length, or between 7 to 80 nucleotides in length, or bebetween 7 to 70 nucleotides in length, or be between 7 to 60 nucleotidesin length, or be between 7 to 55 nucleotides in length, or between 7 to50 nucleotides in length, or be between 7 to 45 nucleotides in length,or be between 7 to 40 nucleotides in length, or be between 7 to 35nucleotides in length, or be between 7 to 30 nucleotides in length, orbe between 7 to 25 nucleotides in length, or be between 7 to 20nucleotides in length, or between 7 to 15 nucleotides in length, or bebetween 7 to 10 nucleotides in length, be between 10 to 100 nucleotidesin length, or be between 10 to 90 nucleotides in length, or between 10to 80 nucleotides in length, or be between 10 to 70 nucleotides inlength, or be between 10 to 60 nucleotides in length, or be between 10to 55 nucleotides in length, or between 10 to 50 nucleotides in length,or be between 10 to 45 nucleotides in length, or be between 10 to 40nucleotides in length, or be between 10 to 35 nucleotides in length, orbe between 10 to 30 nucleotides in length, or be between 10 to 25nucleotides in length, or be between 10 to 20 nucleotides in length, orbetween 10 to 15 nucleotides in length, or between 12 to 100 nucleotidesin length, or be between 12 to 90 nucleotides in length, or between 12to 80 nucleotides in length, or be between 12 to 70 nucleotides inlength, or be between 12 to 60 nucleotides in length, or be between 12to 55 nucleotides in length, or between 12 to 50 nucleotides in length,or be between 12 to 45 nucleotides in length, or be between 12 to 40nucleotides in length, or be between 12 to 35 nucleotides in length, orbe between 12 to 30 nucleotides in length, or be between 12 to 25nucleotides in length, or be between 12 to 20 nucleotides in length, orbetween 12 to 15 nucleotides in length or between be between 15 to 100nucleotides in length, or be between 15 to 90 nucleotides in length, orbetween 15 to 80 nucleotides in length, or be between 15 to 70nucleotides in length, or be between 15 to 60 nucleotides in length, orbe between 15 to 55 nucleotides in length, or between 15 to 50nucleotides in length, or be between 15 to 45 nucleotides in length, orbe between 15 to 40 nucleotides in length, or be between 15 to 35nucleotides in length, or be between 15 to 30 nucleotides in length, orbe between 15 to 25 nucleotides in length, or be between 15 to 20nucleotides in length.

An exemplary embodiment of this invention is a high specificityhybridization probe. The probe (omega probe) sequence is designed toform one or more omega loops flanked by omega arms when hybridized witha target sequence. FIG. 2 shows an exemplary omega probe 220, whichcomprises of a spacer 201, omega arms 1 through 3 202, 204 and 206, andomega loop 1 203 and loop 2 205. The spacer 201 is optional depending onspecific applications and can be attached to either 3′ or 5′ end of theprobe. For applications requiring the attachment of the probe to asurface, it is often preferred to have the attachment through thespacer. In some embodiments, the spacer 201 comprises one or morenucleotides. In some embodiments, the spacer 201 comprises one or morenon-nucleotide moieties. In some embodiments, the non-nucleotidemoieties include but not limited to at least one C3 alkyl spacer, atleast one ethylene glycol spacer, and at least one 1′,2′-dideoxyribose.In some embodiments, omega arm 202, 204, or 206 comprises at least onenucleotide. In some embodiments, the number of nucleotides is between 1and 100. In some embodiments, the number of nucleotides is between 3 and60. In some embodiments, the number of nucleotides is between 5 and 40.In some embodiments, each loop 203 or 205 comprises one or morenucleotides. In some embodiments, the number of nucleotides is between 1and 100. In some embodiments, the number of nucleotides is between 3 and60. In some embodiments, the number of nucleotides is between 5 and 40.In some embodiments, each loop 203 or 205 comprises one or morenon-nucleotide moieties. In some embodiments, the non-nucleotidemoieties include but not limited to at least one C3 alkyl spacer, atleast one ethylene glycol spacer, and at least one 1′,2′-dideoxyribose.In some embodiments, the length of each non-nucleotide loop 203 or 205is between 1 and 200 molecular bonds. In some embodiments, the length ofeach loop 203 or 205 is between 5 and 100 molecular bonds. In someembodiments, the length of each loop 203 or 205 is between 5 and 60molecular bonds.

Thermodynamically, in a binding reaction between the probe 210 and thecorresponding target template sequence 220, the omega arms 202, 204, and206 bind to the template 220, reduce free energy, and stabilize thebinding while the omega loops 203 and 205 are strained, increase freeenergy, and destabilize the binding. In an exemplary embodiment, thelengths of the arms and loops are designed in such a way that the freeenergy decrease due to the arms and the increase due to the loops arecarefully balanced to produce stable omega shaped structures at apredetermined hybridization condition when the probe is hybridized withthe intended target. When the target contains one or more variantnucleotides the probe-target binding of the original structure is nolonger stable and results in reduced binding. A significant advantage ofthe disclosed omega probe design over a regular hybridization probedesign that contains one continuous target-complementary binding segmentis the ability to extend probe length without sacrificing specificity.In general, a nucleic acid binding assay reports the number of targetshybridized to corresponding probes. The number is usually measuredthrough binding densities either on a probe containing surface or in aprobe containing solution. An equilibrium binding density is determinedby binding free energy. The lower the free energy the higher theequilibrium binding density will be (Miura el al. (2005) “A novelstrategy to design highly specific PCR primers based on the stabilityand uniqueness of 3′-end subsequences” Bioinformatics 21 4363-4370). Thebinding free energy of a regular probe increases with the increase ofprobe length. When the regular probe is long (e.g. longer than 35nucleotides), a short variant such as a SNP in the target would onlyproduce a small fraction of free energy increase in the probe-targetbinding. Therefore, sequence specificity of a regular probe decreaseswith increase of probe length. By comparison, free energy of the omegaprobe-target binding is modulated by omega loops and can be maintainedby design at a certain desired level even when the overall bindinglength is long. Therefore, an omega probe retains high specificity overa wider range of probe-target binding length. The free energy of theentire omega structure should kept below zero at the hybridizationcondition. The free energy calculations for the omega primer aredescribed in section “Computation methods” of this specification. Oneexemplary embodiment of the omega probe is microarray assay which mayinvolves one or more detection methods including but not limited tolabeling target sequence with a fluorescence dyes for regularfluorescence detection and labeling omega probes on or near loopnucleotides with donor and acceptor fluorophore groups for FRET(fluorescence resonance energy transfer) detection. These and additionallabeling and detection methods are described by Buzdin in A. Buzdin andS. Lukyanov (2007) “Stem-loop oligonucleotides as hybridization probesand their practical use in molecular biology and biomedicine” NucleicAcids Hybridization, Chapter 14, 85-96, Springer.

Relay PCR

One aspect of the invention relates to a method of target amplificationwhich is named Relay PCR. A complete PCR run consists of twofunctionally distinct but sequentially connected reaction phases, namelytarget selection and library amplification, which are shown in FIG. 3 asphase 1 and phase 2 reactions. A significant advantage of the methods ofthe present invention is that the two functionally distinct reactionphases are carried out in a single tube, in a programmed process on aPCR machine, and without any hands-on operation during the process. Areaction mixture for the relay PCR includes two sets of primers,templates, plus appropriate polymerase, nucleotides and PCR buffer. Thefirst set of primers includes at least one pair of specific primers 301and 302. Generally, each pair of primers delineates a target region 304of interest. An exemplary specific primer pair consists of specificprimer 1 301 and specific primer 2 302. An exemplary specific primer 340(a “regular” primer) consists of a specific segment 341 at 3′ end(between 10 and 80 nucleotides, preferably 12 to 50, in length) and acommon segment 342 at 5′ end (between 10 and 80 nucleotides, preferably12 to 50, in length). The sequence of the specific segment of eachspecific primer is substantially identical or complementary tocorresponding portion of the corresponding target sequence or template.The second set of primers includes at least one pair of common primers.In an exemplary embodiment, one pair of common primers, comprisingcommon primer 1 321 and common primer 2 322, are used. An exemplarycommon primer 350 has a common segment 351 at 3′ end and a tail segmentat 5′ end 352. The sequences of the common segments of specific primer 1301 and common primer 1 321 are substantially the same. The sequences ofthe common segments of specific primer 2 302 and common primer 2 322 aresubstantially the same. In preferred embodiments, the sequences of thecommon segments do not hybridize substantially with any portion ofsample DNA sequences. In preferred embodiments, the concentration of acommon primer is substantially higher than the concentration ofcorresponding specific primers. In some embodiments, the molarconcentration ratio of a common primer to a corresponding specificprimer is at least 50, 100, 500, 1,000, 5,000, 10,000, 50,000, orgreater. Sequence designs of individual primers, suitable concentrationsof the primers, common primer to specific primer concentration ratios,and polymerase selections will become clear as reaction conditions andapplication requirements are described.

FIG. 3 schematically outlines an exemplary embodiment of the relay PCR.In the figure, phase 1 is the target selection phase that consists oftwo thermo cycles and involves a pair of specific primers: specificprimer 1 301 and specific primer 2 302. In cycle 1, specific primer 1301 and specific primer 2 302 are extended on corresponding templates300 producing two replicated cycle-one product sequences 311 and 312 ofcomplementary strands. Herein, templates are nucleic acid sequences onwhich polymerase extension reactions take place and complementaryreplicates are produced. Throughout this specification, terms“template”, “target sequence”, and “sample DNA” are used interchangeablydepending on the context of descriptions. By the extension reaction, thecommon segments of the specific primers are incorporated into thecycle-one product sequences. In cycle 2, specific primer 1 301 andspecific primer 2 302 are extended on corresponding cycle-one productsequences 311 and 312 producing two replicated cycle-two productsequences 323 and 324 of complementary strands. Cycle-two productsequences 323 and 324 are flanked with common segments 325 and 326 ofspecific primers 1 and 2 at 5′ ends and with complementary commonsegments 327 and 328 of the specific primers at 3′ ends. The cycle-twoproduct sequences are the both-end flanked target sequences that areamplified in the remaining thermo cycles. Generally, common primers aredesigned not to hybridize substantially with any portion of sample DNAsequences and not to involve in cycle 1 and cycle 2 reactions. In FIG.3, cycle 3 through cycle N constitute phase 2, in which a library oftarget sequences is amplified. Starting from cycle 3, the both-endflanked target sequences 323 and 324 become available and they carrycomplementary common segments 327 and 328 with which the common primers321 and 322 hybridize and carry out amplification reactions. Whenplurality pairs of specific primers and one pair of common primers areinvolved, all corresponding target sequences are flanked with the samepair of common segments at the end of cycle 2 and are amplified with onepair of common primers between cycle 3 to cycle N (the last cycle). Thehigh concentration ratio of common primer to specific primer ensures thedominance of the common primers in phase 2 amplification reactions.

One aspect of the disclosed relay PCR method is the elimination of theneed to perform multiple hands-on PCR rounds and product purificationsthat were used previously to switch from target specific primeramplifications to common primer amplifications (e.g. Z. Lin et al.(1996), in “Multiplex genotype determination at a large number of geneloci” Proc. Natl. Acad. Sci. 93: 2582-2587; K. E. Varley et al. (2008)“Nested Patch PCR enables highly multiplexed mutation discovery incandidate Genes” Genome Res. 18:1844-1850; and J. Leamon et al. (2012)“Methods and compositions for multiplex PCR” US Patent ApplicationPublication US 2012/0295819 A1).

Another aspect of the disclosed relay PCR method is the exclusion oftarget specific primers from participating in amplification process(cycle 3 to cycle N of FIG. 3) and so as to minimize specific primerrelated amplification bias. A characteristic of the disclosed relay PCRreaction is to limit the function of specific primers to producingflanked target sequences directly from original sample templates inphase 1 and to minimize the contribution of the specific primers to thetarget sequence amplification reactions in phase 2. This minimizes theprimer-dependent amplification yield variations when two or more pairsof specific primers are involved.

Reaction conditions and primer designs have significant impact to theoutcomes of the disclosed relay PCR reactions. For multiplex PCRapplications, a distinct reaction condition of the present invention isthe significantly lower specific target primer concentrations than thatused in regular PCR reactions and in known variations of multiplex PCRreactions. Relay PCR reactions may be performed at much lower specificprimer concentrations than previously used. In regular PCR reactions theprimer concentration is between 100 nM and 5,000 nM (see Dieffenbach etal., PCR Primer: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 2003). In known variations of multiplex PCR reactions, targetspecific primer concentration is between 10 nM and 400 nM (see Henegariuet al. (1997) “Multiplex PCR: Critical Parameters and Step-by-StepProtocol” BioTechniques 23: 504-511; J. Brownie et al. (1997), in “theelimination of primer-dimer accumulation in PCR” Nucleic Acids Res. 25:3235-3241; K. E. Varley et al. (2008) “Nested Patch PCR enables highlymultiplexed mutation discovery in candidate Genes” Genome Res.18:1844-1850; and B. Frey et al. (2013) “Methods and amplification oftarget nucleic acids using a multi-primer approach” US PatentApplication Publication US 2013/00045894 A1). By comparison, in anexemplary embodiment of the present invention, as shown in Examples I toIII, the concentration of each individual specific primer is 1 nM orlower and the concentration of each common primer is 500 nM. Anotherdistinct reaction condition of the present invention is the use ofsignificantly extended annealing times in thermo cycles 1 and 2 incombination with the low specific primer concentrations. In regular PCRand known variations of multiplex PCR reactions, annealing time isbetween 10 sec and 2 min. By comparison, in an exemplary embodiment ofthe present invention, as shown in Examples I through IV, extensiontimes between 30 min and 4 hours are used in thermo cycles 1 and 2.Using the previously reported annealing time of 30 sec (e.g. B. Frey etal. (2013) “Methods and amplification of target nucleic acids using amulti-primer approach” US Patent Application Publication US2013/00045894 A1) at specific primer concentration of 1 nM or lower theamount PCR product obtained was low and was below the detection limit ofregular agarose gel electrophoresis measurement in experiments. Anotherdistinct reaction condition of the present invention is the use of highannealing temperatures in cycles 1 and 2, in combination with the longextension times. These exemplary reaction conditions merely representcertain aspects of the present invention and are not intended, norshould be construed, as limiting the invention in any manner. The exactreaction conditions for specific applications can be determined by thosewho are skilled in the art by following the teachings of thisspecification in whole including references.

One aspect of the present invention is the use of omega primersadvantageously as specific primers in relay PCR. FIG. 4A schematicallyoutlines exemplary embodiments of a relay PCR using omega primers 401and 402 as specific primers. In each primer, 3p arm 403 and 5p arm 405are both target sequence specific. In other words, the arm sequences arecomplementary to the priming sections of corresponding templatesequences 400. Loop sections 404 and 441 of the omega primers serve ascommon segments of the specific primers. Common primers 421, 422, and445 consist of common segments 446 at 3′ end and tail segments 447 at 5′end. The sequences of the common segments 441 and 446 of correspondingcommon primers 440 and omega primers 445 are substantially the same.

As shown in FIG. 4A, a complete relay PCR process comprises two phases,phase 1 for target selection and phase 2 for amplification. The targetselection phase consists of two thermo cycles and involves a pair ofspecific primers: specific primer 1 401 and specific primer 2 402. Incycle 1, specific primer 1 401 and specific primer 2 402 are extended oncorresponding templates 400 producing two replicated cycle-one productsequences 410 of complementary strands. The original specific primer 402comprises the 5′ section of the cycle-one products 410. The original 3parm 403, loop 404, and 3 p arm 405 of specific primer 402 becomespecific priming segment 411, common priming segment 412, and 5p armtail segment 413 of the cycle-one product 410. In cycle 2, specificprimer 1 401 and specific primer 2 402 are extended on correspondingcycle-one product sequences 410 producing two replicated cycle-twoproduct sequences 420 of complementary strands. Cycle-two productsequences 420 are flanked with common segments 412 and 414 in sectionsclose to 5′ ends and with complementary common segments 423 and 424 insections close to 3′ ends. The complementary common segments 423 and 424are complementary to common primers 421 and 422 and serve as primingsites for PCR amplification reaction from cycle 3 to cycle N. In someembodiments involving plurality pairs of specific primers, all primerpairs share the same pair of loop sequences so that a library of targetsequences are amplified by one pair of common primers. The 5p arm tails413 of cycle-one and cycle-two products are bypassed for libraryamplification by the common primers 422 and 423. As result, the finalamplification products 430 retain only 3p arm portions of the originalomega specific primers.

While omega primers in FIG. 4A have internal loops, omega primers ofother loop forms can be used in relay PCR as well. As another exemplaryembodiment, FIG. 4B depicts a relay PCR process using omega primers ofbulge loops as specific primers 451 and 452. The target selection andlibrary amplification principles of the processes shown in FIG. 4A andFIG. 4B are substantially the same.

An exemplary application of combining omega primers with relay PCR istarget enrichment for sequencing assay. The combination bringssignificant and unique benefits to the application. The fact that onlythe 3p arm portions 403 and 453 of the original omega specific primersare retained in the amplification products is highly desirable since itprovides an opportunity to minimize the lengths of specific primersections 431 and 481 in the amplified target sequences 430 and 480. Thisis achieved by using long 5p arms and short 3p arms while maintainingsufficiently stable bindings between the omega primers and correspondingtemplates at corresponding annealing temperature. For sequencing use,sequencing reads from the native sections 432 and 482 between specificpriming sections 431 or 481 are from the native sequences of testingsamples while sequencing reads from the specific primer sections 431 and481 are dictated by the primers used. Since the reading length of asequencing run is limited, it is highly desirable to shorten thespecific primer sections 431 and 481 so as to maximize the usefulreading length of the native sequences. In some embodiment, the tailsegment 352, 447 and 497 of common primer 1 further comprises at leastone barcode section. In some embodiment, the tail segment 352, 447 and497 of common primer 2 further comprises at least one barcode section.In some embodiment, the tail segments 352, 447 and 497 of both commonprimer 1 and common primer 2 further comprise at least one barcodesection. The design and the use of the barcodes are described inBystrykh L V (2012) Generalized DNA Barcode Design Based on HammingCodes. PLoS ONE 7(5): e36852. doi:10.1371/journal.pone.0036852.

In some embodiment, at least one omega primer contains two or moreloops. In one exemplary embodiment shown in FIG. 5, a specific primer501 comprises of a relay priming loop 502 and an insertion loop 503. Therelay priming loop 502 serves as common segment of the specific primer.In cycle 1 reaction specific primer 1 501 extends and form cycle 1product 511. This results in the incorporation of the insertion loop 503into product as an insert 513. The remaining cycles proceed in a similarfashion as described above relating to the relay PCR processes of FIG.4A and FIG. 4B. At the completion of the process, an insert 533 isincorporated into the final product 530. Exemplary applications of suchsegmented primers include but not limited to mutagenesis, gene knockout, gene knock in, signature tag, protein engineering, and genetherapy.

Some embodiments of target enrichment using relay PCR comprise targetselection using singular primer extension and amplification using commonprimer PCR: singular primer extension relay PCR. FIG. 6 depicts anexemplary application embodiment, which comprises sample preparation andthe relay PCR processes. The sample preparation involves fragmentationand adapter ligation. The process is well known to the field ofmolecular biology and is described in detail by N. Arneson et al. (2008)“Whole-Genome Amplification by Adaptor-Ligation PCR of Randomly ShearedGenomic DNA (PRSG)” Cold Spring Harb Protoc; and by D. Bentley et al.(2008) “Accurate whole human genome sequencing using reversibleterminator chemistry” Nature 456 53-59 and associate supportingdocuments. An exemplary sample preparation starts with randomfragmentation of double stranded DNA template 600 into short segments.The double stranded DNA sequences can be produced from various sourcesincluding not limited to genomic DNA and RNA derived double strandedcDNA. Fragmentation of the double stranded DNA can be accomplished usingone or more various well known processes including nebulization,sonication, and enzymatic digestion. Nebulization may be accomplishedusing a commercial product Nebulizers and product instructions from LifeTechnologies (Grand Island, N.Y.). Sonication may be accomplished usingone of various commercial products, e.g. Focused-ultrasonicator fromCovaris instrument (Woburn, Mass.). Enzymatic digestion may beaccomplished using commercial kit NEBNext® dsDNA Fragmentase from NEB(Ipswich, Mass.). Repair the ends of the DNA fragments using NEBNext®Ultra™ End Repair/dA-Tailing Module from NEB (Ipswich, Mass.). Addadaptors 603 to the end polished fragments by ligation using T4 DNAligase from NEB (Ipswich, Mass.). Adaptor flanked fragments 604 areobtained. Adaptor 603 is a double strand DNA sequence comprising a plusstrand oligonucleotide 601 and a minus strand oligonucleotide 602. Insome embodiments, plus strand oligonucleotide 601 and minus strandoligonucleotide 602 are completely complementary to each other. In someembodiments, plus strand oligonucleotide 601 and minus strandoligonucleotide 602 are partially complementary to each other. In someembodiments, plus strand oligonucleotide 601 is shorter than minusstrand oligonucleotide 602 and is substantially complementary to 3′section of the minus strand oligonucleotide 602. In some embodiments,the 3′ end of plus strand oligonucleotide 601 a modified nucleotide thatblocks the oligonucleotide from polymerase extension reaction. Theexemplary modified nucleotide includes but is not limited todideoxycytidine, inverted dT, 3′ amino modifier, and 3′ biotin. In someembodiments, 5′ end of the plus strand oligonucleotide 601 isphosphorylated. In some embodiments, 3′ end of the minus strandoligonucleotide 602 is a dA overhang.

The lower portion of FIG. 6 depicts the singular primer extension relayPCR process in which each target sequence is selected by the extensionreaction of a singular specific primer and then amplified by PCR of onepair of common primers. In the exemplary embodiment, the reactionmixture comprises adaptor flanked fragments 604 as sample templates, oneor more omega primers as target specific primers 610, common primer 1611, common primer 2 612, and polymerase, all mixed in a PCR buffersolution. Each omega primer comprises a 3p arm 615, at least one loop616, and a 5p arm 617. The sequences of 3p arm 615 and 5p arm 617 aredesigned based on predetermined target sequences from the starting DNAtemplate 600. The loop 616 contains a section having substantially thesame sequence as 3′ section of common primer 2 612. The sequences of 3′section of common primer 1 611 and a selected section of minus strandoligonucleotide 602 are substantially the same. In some embodiments, theselected section of minus strand oligonucleotide 602 covers asubstantial portion of minus strand oligonucleotide 602. In someembodiments, the selected section of minus strand oligonucleotide 602covers a substantial portion of minus strand oligonucleotide 602 minusthe portion overlapping with plus strand oligonucleotide 601. Incycle-one reaction, specific primer 610 binds to the correspondingtarget sequence fragment 614 and then is extended to produce cycle-oneproduct 620. In cycle-two, common primer 1 611 binds to the adaptor 621section of the cycle-one product 620 and then extends to producecycle-two product 630. The cycle-two product 630 contains a commonpriming section 631 that is complementary to loop 616 of specific primer610 and binds with common primer 2 612 to facilitate PCR amplificationin the remaining cycles. From cycle 3 to cycle N the specific primer 610selected target sequences are amplified by the paired common primer 1611 and common primer 2 612 resulting in product 640. While theexemplary embodiment of FIG. 6 depicts omega primers as the specificprimers, alternative embodiments may use regular primers as the specificprimers in the singular primer extension relay PCR. The use of regularprimers in relay PCR has been described above relating to FIG. 3. Aregular primer is one that is not an omega primer; a regular primer hasa binding segment without loops and/or, in some embodiments, extraneousmismatches (i.e., it has a one-to-one correspondence with its targetsequence).

Separation

In some embodiments, the relay PCR product solutions are furtherpurified to separate the PCR products 330, 430, 480, 530, and 640 fromthe rest of the reaction mixture. Various established PCR reactionpurification methods and commercial kits can be used for the purpose.These include but not limited to normalization beads from AxygenBiosciences (Union City, Calif.), PCR purification columns from Qiagen(Valencia, Calif.), and gel cut purification. FIG. 7 illustrates anexemplary embodiment of a purification method of this invention. Thestarting mixture comprises target sequences 700, common primer 1 (701),common primer 2, fragments 707, and polymerase, all in a PCR buffersolution. The target sequences 700 are flanked with priming segmentsthat are either identical or complementary to common primer 1 and commonprimer 2 and are designed to be amplified by the primers. Fragments 707do not carry the priming segments and are not expected to be amplifiedby the primers. This starting mixture is similar to the reaction mixtureof FIG. 6 at the end of the cycle 1 reaction. Common primer 2 (702)comprises of a priming segment, a modifier segment 704 and a tag segment705. The function of the modifier segment 704 is to prohibit thepolymerase extension reaction from passing through the segment and/or tofacilitate enzymatic, chemical, or photo cleavage at the location.Exemplary embodiments of modifier segment 704 include but not limited toat least one C3 alkyl spacer, at least one ethylene glycol spacer, atleast one photo-cleavable spacer, at least one 1′,2′-dideoxyribose, andat least one deoxyuridine. The incorporations of these modifiers intooligonucleotides are well known in the field of nucleic acid synthesisand can be performed by commercial suppliers such as Integrated DNATechnologies, Inc. (Coralville, Iowa). In an exemplary embodiment,common primer 2 (702) comprises a hexa-ethylene glycol spacer as themodifier segment 704 that stops polymerase extension reaction frompassing through the segment and into tag segment 705. This results inPCR product 710 containing a single strand overhang tag segment 715. Insome embodiments the tag segment 705 comprises an oligonucleotide. Insome embodiments the tag segment 705 comprises of at least one bindingmoiety. An exemplary binding moiety is biotin. In some embodiments thetag segment 705 comprises of a combination of an oligonucleotide and abinding moiety. In some embodiments the binding moiety is attached tothe 5′ end of the tag segment 705 oligonucleotide. In some embodiments,one or more of the above mentioned purification kits or methods areapplied to the PCR product solution to remove polymerase and residualsingle stranded primers. In some applications, such as singular primerextension relay PCR, fragments 717 have an average size that is similarto that of PCR product 710 and an additional or a different purificationprocess is needed to remove fragments 717.

The lower portion of FIG. 7 illustrates an exemplary embodiment of thedisclosed purification method involving the direct capture of the PCRproduct 720. Add probe 722 grafted beads 721 into the PCR productsolution or a primer removed PCR product solution. Probe 722 has asequence that is substantially complementary to that of tag segment 715and facilitates the capture of the PCR product 720 by the beads 721through hybridization. Optionally, the solution salt concentration maybe adjusted to ensure a sufficient hybridization. Salts include but arenot limited to sodium chloride, sodium citrate, sodium phosphate,potassium chloride, potassium phosphate, ammonium chloride, trischloride, and/or mixture of two or more salts. Agitation and incubationof the solution at room temperature or at a predetermined temperatureincrease the capture yield. Washing the beads 721 with a hybridizationbuffer solution removes fragments 717, residual polymerase and freeprimers while retaining the hybridized PCR products 720. To complete thepurification, the beads 721 are placed into an elution buffer and thebuffer temperature is elevated to release the PCR product 720 from thebeads 721. The immobilization of oligonucleotides to form probe 722grafted beads 721 and the use of the beads to capture nucleic sequencesare well known in the field of biology. An exemplary embodiment usesDynabeads® MyOne™ Streptavidin C1 from Life Technologies (Grand Island,N.Y.) as beads 721. Sequence designs for the complementary pair of probe722 and tag segment 705 share many principles and procedures of thehybridization probe designs that are familiar to one skilled in the art.The sequences are designed in such way that a stable hybridizationbetween the pair is achieved during binding process and in thehybridization buffer but denaturation between the pair is achievedduring release process. In general, the binding and/or hybridizationbuffers have a relatively high salt concentration of at least 50 mM, 100mM, 500 mM, 1M, 2M, or higher. The elution buffer has a relatively lowsalt concentration of at most 100 mM, 50 mM, 10 mM, 5 mM, 1 mM or lower.In general, the binding temperature is lower than elution temperature.The temperatures are decided based on Tm (melting temperature) of theprobe 722/tag 705 pair in the corresponding buffer solutions. Apreferred binding temperature is below the Tm of the probe/tag pair inthe binding or hybridization buffer. A preferred elution temperature isabove the Tm of the probe/tag pair in the elution buffer. In someembodiments, the PCR products 730 remain double stranded after theelution process. In some embodiments, binding temperature is at most 40°C., 35° C., 30° C., 25° C., 22° C., or lower. Elution temperature is atleast 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., or higher. Otherconsiderations in the sequence design include the minimizing crosshybridization of the probe 722 sequence to any target sequences andminimizing secondary structure involving tag segment 705.

Alternative embodiments of the disclosed method involve indirect captureof the PCR products 720. In some embodiments, probes 722 are notpre-grafted to beads 721 but rather contain ligand moieties, such asbiotin. The beads 721 are coated with capture moieties such asstreptavidin. In the embodiments, first, the free probes 722 arehybridized with the tag segments 715. Then the capture moiety coatedbeads 721 are added to the hybridization solution to capture the PCRproduct 720. The remaining processes are similar to what describedabove.

One aspect of the above described purification method is the directformation of PCR products with single-strand overhangs. The overhangsmay be used to facilitate purification. The method makes it possible torelease the double stranded product 730 in a mild denaturationcondition. By comparison, a regular biotinylated primer, without themodifier segment 704 and tag segment 705, would produce blunt ends.Although the PCR product can be captured by streptavidin coated beads,the release of the product requires strong denature conditions (see usermanual of Dynabeads® MyOne™ Streptavidin C1 from Life Technologies,Grand Island, N.Y.) under which the double stranded structure of the PCRproduct would be denatured as well.

This disclosure describes the use of the single strand overhangcontaining PCR products in new and improved approaches for librarypreparation and cluster formation in surface cluster based sequencing(D. Bentley et al. (2008) “Accurate whole human genome sequencing usingreversible terminator chemistry” Nature 456 53-59). FIG. 8 schematicallyillustrates the new approaches. The top of FIG. 8 illustrates targetamplification by PCR, which is similar to that of FIG. 7 and has beendescribed above. The target amplification process shown in FIG. 8 is aPCR process for general use, including for sequence library preparationin Next Generation Sequencing (Mardis ER (2008) “Next-generation DNAsequencing methods”, Annu Rev Genomics Hum Genet 9: 387-402). Onefeature of the disclosed method is to produce PCR products 810 thatcontain single-strand tag 815. In some embodiments, the common primer 2(802) comprising priming segment 803, modifier segment 804 and tagsegment 805 is used as common primer 1 321, 421, 471, 521, and 611 or ascommon primer 2 322, 422, 472, 523, and 612 in relay PCR processes ofFIGS. 3 through 6. The disclosed method further comprises immobilizationof the single-strand tag 815 containing PCR products 810 to a substratefor conducting chemical and/or biochemical reactions. As an exemplaryillustration, in FIG. 8, a substrate 826 is grafted with probe 822,surface primer 1 (823), and surface primer 2 (824). Depending onapplications, the substrate 826 can be made of glass, silicon, polymer,metal or any other appropriate materials. The grafted moieties, probe822, surface primer 1 (823), and surface primer 2 (824) areoligonucleotides of predetermined sequences. In some embodiments, thegrafted moieties further comprise spacer 827 to connect theoligonucleotides to substrate surface. Grafting oligonucleotides tosubstrate surfaces is a well-known art and has been described inliterature (B. Joos et al. (1997) “Covalent attachment of hybridizableoligonucleotides to glass supports” Anal Biochem, 247 96-101; Y. Rogerset al. (1999) “Immobilization of oligonucleotides onto a glass supportvia disulfide bonds: A method for preparation of DNA microarrays” AnalBiochem, 266 23-30; D. Bentley et al. (2008) “Accurate whole humangenome sequencing using reversible terminator chemistry” Nature 45653-59, Supplementary Information). Immobilization of the PCR products810 begins with co-hybridization of single-strand tag 815, guide 821,and probe 822. Guide 821 is an oligonucleotide having a portion of thesequence complementary to tag 815 and a portion of the sequencecomplementary to probe 822. In some embodiments, the co-hybridization isperformed by first applying a guide 821 solution to substrate 826 at asufficiently high guide 821 concentration to substantially hybridize allprobes 822 on the substrate and then wash away any extra guide 821 inthe solution. This step produces guide/probe pairs on the substratesurface. The co-hybridization is completed by applying a PCR product 810solution at a sufficiently high PCR product 810 concentration (thresholdconcentration) to substantially hybridize all the guide/probe pairs. Insome embodiments, upon the hybridization, 5′ end of tag 815 stacks to 3′end of the probe and there is no gap in between the two ends. In someembodiments, the 5′ terminal of tag 815 is phosphorylated and 3′terminal of probe 822 is a hydroxyl group. Tag 815 and probe 822 may becovalently joined together by ligation. In some embodiments, theligation is done using T4 ligase from NEB (Ipswich, Mass.).

The main considerations for sequence designs of tag 815, probe 822, andguide 821 include melting temperature, sequence uniqueness, andintra-molecular folding. Melting temperatures for both tag/guide andprobe/guide pairs should be considerably above both hybridization andligation temperatures in corresponding buffers so that sufficiently highhybridization yields can be achieved and/or maintained in both reactionconditions. In some embodiments, melting temperatures of probe/guidepair is higher than that of tag/guide pair. All three sequences shouldhave minimal folding under the reaction conditions. All three sequencesshould be sufficiently different from that of surface primer 1 (823) andsurface primer 2 (824) so that no cross hybridization to the two surfaceprimers will take place. Additionally, hybridization temperature,ligation temperature and buffer compositions should be designed in suchway that the double stranded structure of PCR product 810 remains stablein both reactions. More accurate sequence and reaction condition designswill be described in later sections of this disclosure.

Alternative methods can be used to join tag 815 and probe 822. In someembodiments, the 5′ terminal of tag 815 is an azide group and 3′terminal of probe 822 is an alkyne group. Tag 815 and probe 822 are thenjoined together using click chemistry as described by R. Kumar el al.(2007) “Template-Directed Oligonucleotide Strand Ligation, CovalentIntramolecular DNA Circularization and Catenation Using Click Chemistry”J. AM. CHEM. SOC. 129, 6859-6864.

In some embodiments, after the tag 815 and the probe 822 are covalentlyjoined, the system, including substrate and immobilized PCR product 820,is placed into a denature buffer and is washed to remove plus strand 828and guide 821. In some embodiments, the denature buffer comprises highconcentration of formamide. The result is a single strand product 830covalently attached to substrate 826.

In some embodiments, product 830, surface primer 1 (823), and surfaceprimer 2 (824) are subject to further enzymatic and/or chemicalreactions to extend the surface primers using the product 830 as initialtemplate thereby producing additional copies of product 830 in thevicinity of the starting product 830 which form surface clusters of theproduct 830. The parallel copies of product 830 are then used astemplates for sequencing. The surface cluster formation and thesequencing process have been described in detail in D. Bentley et al.(2008) “Accurate whole human genome sequencing using reversibleterminator chemistry” Nature 456 53-59 and associated supplementaryinformation which are hereby incorporated by reference in theirentirety. In some embodiments, the sequence of surface primer 1 (823) issubstantially complementary to the sequence of flank 1 (831). In someembodiments, the sequence of surface primer 2 (824) is substantially thesame as the sequence of flank 2 (833). In some embodiments, the surfacedensity of surface primer 1 (823) is substantially the same as thesurface density of surface primer 2 (824). The definition of the surfacedensity is the number of immobilized molecules per unit surface area. Insome embodiments, surface densities of surface primer 1 (823) andsurface primer 2 (824) are substantially higher than that of probe 822.In some embodiments, the surface density ratio of surface primer 1 (823)(or surface primer 2 (824)) to probe 822 is at least 1,000, 10,000,100,000, 1,000,000, 10,000,000, 100,000,000, or greater. In someembodiments, the surface density ratios are controlled by relativeconcentrations of probe 822, primer 1 (823), and primer 2 (824) mixturesolution for the grafting preparation of substrate surface.

Certain aspects of the disclosed method include the addition of probesas well as surface primers to the substrate surface and the use of probecaptured sequences as the seeds of cluster formation. In the state ofart practice (D. Bentley et al. (2008) “Accurate whole human genomesequencing using reversible terminator chemistry” Nature 456 53-59), thesubstrate surface may be grafted with only two primers which are presentin substantially equal surface densities. The seeding of surfaceclusters is formed by hybridization between one of the surface primersand one of the flanks of PCR product followed by polymerase extension ofthe primer. While the surface primers are densely populated on thesubstrate surface, the seed density is controlled by the solutionconcentration of the PCR product. Under normal conditions, the endcluster density is proportional to the seed density. In a parallelsequencing process, signals emitted from each cluster are used to derivethe sequence of the seed template. In order to obtain reliable signaldetection from individual clusters, the distance between adjacentclusters needs to be sufficient, otherwise, signals emitted fromadjacent clusters would be inseparable and erroneous sequence readswould be produced. On the other hand, for the purpose of maximizing readthroughput it is desirable to increase the cluster density. There is anoptimal cluster density. In current practice the optical cluster densityis achieved by carefully controlling the PCR product concentration inthe clustering solution. The process is time consuming and subjects tomeasurement instrument and human handling errors. By comparison, in thepresent method, cluster density is controlled by probe density orsurface primer to probe ratio which are fixed during substrate surfacepreparation by the substrate manufacturer. As result, for the clusterpreparation at user's site, as long as the user makes the PCR productconcentration beyond a threshold the resulted cluster density willalways stay near a fixed value.

In some embodiments, multiple probes 822 of distinct sequences are used.This arrangement is particularly useful when multiple samples aresequenced in parallel. Target amplification is applied on each samplewith a unique pair of common primer 1 (801) and common primer 2 (802).The common primers 2 (802) for different samples comprise different tags805 of distinct sequences. Each unique tag 805 is paired with a uniqueprobe 822 through a unique guide 821. The design considerations of themultiple tags, probes, and guides must include the minimization ofcross-hybridization among the sequences. In some embodiments, the numberof distinct probes is at least 1, 2, 4, 8, 12, 16, 32, 48, 64, 96, ormore. In some embodiments, the surface density of each distinct probe issubstantially the same for all probes. In some embodiments, the surfacedensities of different probes are different. In some embodiments, thesurface densities of a selected group of probes are set at predeterminedvalues while the surface densities of the rest of the probes are madesubstantially equal. In some embodiments, the surface density ratio ofsurface primer 1 (823) (or surface primer 2 (824)) to the combinedprobes 822 is at least 1,000, 10,000, 100,000, 1,000,000, 10,000,000,100,000,000, or greater. In some embodiments, common primer 1 (801)comprises at least one barcode section. In some embodiment, the primingsegments of common primer 2 (802) comprises at least one barcodesection. The combination of the barcodes in the primers is used toidentify the origin of the sample in sequence reads. In an exemplaryembodiment of using the multi-probe containing substrate in clusterpreparation for multi-sample parallel sequencing, a solution mixture ofall involving guides 821 to the substrate 826 is applied to formguide/probe pairs. Any extra guides 821 are then washed away. Then, asolution mixture of tag 815 containing PCR products 810 of all samplesis applied to the substrate to complete tag/guide/probeco-hybridization. Ligation, denaturation, and cluster formation areconducted as described above. As result, the number of clustersattributed to each sample is directly related to the number of availablesample specific probes on the substrate surface and has little to do thePCR product concentration of the sample, as long as the concentration isabove a threshold value. By comparison, the current state of the artcluster preparation methods for multiple sample parallel sequencingrequires precision sample quantity normalization for all samplesinvolved and precisely controlled total PCR product concentration. Theself-limiting feature of the present methods provide significantadvantages over the current method in terms of ease operation, resultconsistency and overall process robustness.

Polymerase Selection

In some embodiments of the present invention, the amplified products areformed via polymerase chain reaction using one or more DNA polymerases.In some embodiments, the polymerase can be a thermo stable polymerase.In some embodiments, the polymerase can be a hot-start polymerase. Insome embodiments, the polymerase can be a high fidelity polymerase. Insome embodiments, the polymerase can be a recombinant polymerase. Insome embodiments, the polymerase can be a commercially available productsuch as Platinum® Taq DNA Polymerase (Life Technologies, Grand Island,N.Y.), AccuPrime™ Taq DNA Polymerase (Life Technologies, Grand Island,N.Y.), AmpliTaq Gold® DNA Polymerase (Life Technologies, Grand Island,N.Y.), Taq DNA Polymerase (New England Biolabs, Ipswich, Mass.), OneTaq®DNA Polymerase (New England Biolabs, Ipswich, Mass.), Deep Vent™ DNAPolymerase (New England Biolabs, Ipswich, Mass.), Phusion® Hot StartFlex DNA Polymerase (New England Biolabs, Ipswich, Mass.), Q5®High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, Mass.),PfuTurbo Cx Hotstart DNA Polymerase (Agilent, Santa Clara, Calif.),PfuUltra II Fusion HS DNA Polymerase (Agilent, Santa Clara, Calif.),KAPA HiFi PCR Kits (KAPA Biosystems, Wilmington, Mass.).

In some embodiments of the present invention, one or more hot-startpolymerases are advantageously used to minimize potential off-targetamplification and primer-dimer formation.

In some embodiments, a new class of thermo-stable high-fidelitypolymerases that lacks both strand-displacement activity and 5′ to 3′nuclease activity is advantageously used. The lack of stranddisplacement and 5′ to 3′ nuclease activity benefits multiplexamplification applications in which two or more target regions are intandem or in positional proximity. By using these polymerases, primersbinding to the middle of the tandem regions will not be displaced ordegraded. Commercial products of this new class of thermo-stablehigh-fidelity polymerases include, but are not limited to, Phusion® HotStart Flex DNA Polymerase (New England Biolabs, Ipswich, Mass.) and Q5®High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, Mass.).

Reaction Conditions

Reaction conditions are generally governed by polymerases involved,primer thermodynamic properties, target sequence properties, andmultiplexity of a multiplex PCR. A relay PCR run includes twofunctionally different reaction phases, which may require differentreaction conditions. The specific reaction conditions for specificapplications can be determined by those who are skilled in the art byfollowing specific considerations below, examples at the end of thisdescription, and teachings and references throughout of thisspecification.

Phase 1 function of a relay PCR is to produce common segment flankedspecific target sequences. Corresponding reaction conditions should bedesigned to achieve the objectives of producing the highest possibleyields for on-target replications, minimizing off-target templatereplications, and minimizing primer-dimer formation. As described inFIG. 3 through FIG. 5, the phase 1 reaction consists of cycle 1 andcycle 2 reactions. In FIG. 6, the phase 1 reaction comprises cycle 1reaction. Each cycle contains three thermo steps including denaturing,annealing, and extension. The annealing conditions of cycle 1 and cycle2 require critical considerations. The following principles are used toguide the considerations. The probability of primer-dimer formationincreases as the number of specific primers increases (see U. Landegrenet al. (1997) “Locked on target: strategies for future gene diagnostics”Ann. Med., 29: 585-590). On the other hand, based on chemical reactionthermodynamics and kinetics (see I. Tinoco et al. (1995) “PhysicalChemistry: Principles and Applications in Biological Sciences” PrenticeHall College Div; 3rd edition), the equilibrium product concentrationsand the rates of primer-primer as well as on-target primer-templatehybridization interactions decrease as the concentrations of the primersdecrease. Additionally, based on nucleic hybridization thermodynamicprinciples (see J. SantaLucia Jr. et al. (2004) “The thermodynamics ofDNA structural motifs” Annu. Rev. Biophys. Biomol. Struct. 33:415-440),hybridization stability increases as hybridization temperature decreasesand complementary sequence length increases. In general, as the plexityof an amplification reaction increases and/or as the number of specificprimers increases, concentration of each individual primer should bedecreased to reduce the probability of primer-dimer formation; at thesame time the primer concentrations should not be decreased sodramatically that the desired interactions between the primers andtemplates are not reduced. In such cases where the concentration of theprimers are reduced, the annealing time should be increased tocompensate for the reduced interaction rate between the primers and thetemplates. High annealing temperature is generally preferred forobtaining high primer specificity and for minimizing undesirableprimer-primer hybridization. The high annealing temperature is alsopreferred to avoid a net degradation of the primer during a longannealing period when the selected polymerase has a high 3′ to 5′nuclease activity. The optimal annealing temperature is generallyexpected to be found close to the peak polymerase activity temperaturewithin a 10° C. range. The peak polymerase activity temperature can beobtained from corresponding polymerase suppliers.

Exemplary reaction conditions of the present invention are provided inExample I through IV of this description. For high plexity amplificationapplications, optimal reaction conditions vary significantly frompreviously known conditions. Target specific primer concentrations are0.001 nM or lower, 0.01 nM or lower, 0.1 nM or lower, 1 nM or lower, 2nM or lower, 3 nM or lower, 4 nM or lower or 5 nM or lower. In certainembodiments of the present invention the target specific primerconcentrations are from about 0.0001 nM to about 10 nM or from about0.0001 nM to about 5 nM or from about 0.0001 nM to about 4 nM or fromabout 0.0001 nM to about 3 nM or from about 0.0001 nM to about 2 nM orfrom about 0.0001 nm to about 1 nM or from about 0.0001 nm to about 0.1nM or from about 0.0001 nm to about 0.01 nM or from about 0.0001 nm toabout 0.001 nM, or from about 0.001 nM to about 10 nM or from about0.001 nM to about 5 nM or from about 0.001 nM to about 4 nM or fromabout 0.001 nM to about 3 nM or from about 0.001 nM to about 2 nM orfrom about 0.001 nm to about 1 nM or from about 0.001 nm to about 0.1 nMor from about 0.001 nm to about 0.01 nM, or from about 0.01 nM to about10 nM or from about 0.01 nM to about 5 nM or from about 0.01 nM to about4 nM or from about 0.01 nM to about 3 nM or from about 0.01 nM to about2 nM or from about 0.01 nM to about 1 nM or from about 0.01 nM to about0.1 nM, or from about 0.1 nM to about 10 nM or from about 0.1 nM toabout 5 nM or from about 0.1 nM to about 4 nM or from about 0.1 nM toabout 3 nM or from about 0.1 nM to about 2 nM or from about 0.1 nM toabout 1 nM, or from about 1 nM to about 10 nM or from about 1 nM toabout 5 nM or from about 1 nM to about 4 nM or from about 1 nM to about3 nM or from about 1 nM to about 2 nM. The annealing time may be greaterthan 5 minutes, greater than 10 minutes, greater than 20 minutes,greater than 30 minutes, greater than 40 minutes, greater than 50minutes, greater than 60 minutes, greater than 70 minutes, greater than80 minutes, greater than 90 minutes, greater than 100 minutes, greaterthan 110 minutes, greater than 120 minutes greater than 130 minutes,greater than 140 minutes, greater than 150 minutes, greater than 160minutes, greater than 170 minutes, greater than 180 minutes greater than190 minutes, greater than 200 minutes, greater than 210 minutes, greaterthan 220 minutes, greater than 230 minutes, greater than 240 minutes.The annealing time may be from about 5 minutes to about 500 minutes, orfrom about 5 minutes to 400 minutes or from about 5 minutes to about 300minutes or from about 5 minutes to about 250 minutes or from about 5minutes to about 200 minutes or from about 5 minutes to about 150minutes or from about 5 minutes to about 100 minutes or from about 5minutes to about 50 minutes, or from about 10 minutes to about 500minutes, or from about 10 minutes to 400 minutes or from about 10minutes to about 300 minutes or from about 10 minutes to about 250minutes or from about 10 minutes to about 200 minutes or from about 10minutes to about 150 minutes or from about 10 minutes to about 100minutes or from about 10 minutes to about 50 minutes, or from about 20minutes to about 500 minutes, or from about 20 minutes to 400 minutes orfrom about 20 minutes to about 300 minutes or from about 20 minutes toabout 250 minutes or from about 20 minutes to about 200 minutes or fromabout 20 minutes to about 150 minutes or from about 20 minutes to about100 minutes or from about 20 minutes to about 50 minutes, or from about30 minutes to about 500 minutes, or from about 30 minutes to 400 minutesor from about 30 minutes to about 300 minutes or from about 30 minutesto about 250 minutes or from about 30 minutes to about 200 minutes orfrom about 30 minutes to about 150 minutes or from about 30 minutes toabout 100 minutes or from about 30 minutes to about 50 minutes or fromabout 40 minutes to about 500 minutes, or from about 40 minutes to 400minutes or from about 40 minutes to about 300 minutes or from about 40minutes to about 250 minutes or from about 40 minutes to about 200minutes or from about 40 minutes to about 150 minutes or from about 40minutes to about 100 minutes or from about 40 minutes to about 50minutes, from about 50 minutes to about 500 minutes, or from about 50minutes to 400 minutes or from about 50 minutes to about 300 minutes orfrom about 50 minutes to about 250 minutes or from about 50 minutes toabout 200 minutes or from about 50 minutes to about 150 minutes or fromabout 50 minutes to about 100 minutes or from about 60 minutes to about500 minutes, or from about 60 minutes to 400 minutes or from about 60minutes to about 300 minutes or from about 60 minutes to about 250minutes or from about 60 minutes to about 200 minutes or from about 60minutes to about 150 minutes or from about 60 minutes to about 100minutes. High annealing temperatures are preferred. The annealingtemperature may be greater than 50° C., or greater than 60° C., orgreater than 65° C., or greater than 70° C., or greater than 75° C., orgreater than 80° C., or greater than 85° C., or greater than 90° C. Theannealing temperature may be from about 50° C. to about 95° C., or fromabout 50° C. to about 90° C., from about 50° C. to about 85° C., fromabout 50° C. to about 80° C., from about 50° C. to about 75° C., fromabout 50° C. to about 70° C., from about 50° C. to about 65° C., fromabout 50° C. to about 60° C., or from about 55° C. to about 95° C., orfrom about 55° C. to about 90° C., from about 55° C. to about 85° C.,from about 55° C. to about 80° C., from about 55° C. to about 75° C.,from about 55° C. to about 70° C., from about 55° C. to about 65° C.,from about 55° C. to about 60° C., or from about 60° C. to about 95° C.,or from about 60° C. to about 90° C., from about 60° C. to about 85° C.,from about 60° C. to about 80° C., from about 60° C. to about 75° C.,from about 60° C. to about 70° C., from about 60° C. to about 65° C., orfrom about 65° C. to about 95° C., or from about 65° C. to about 90° C.,from about 65° C. to about 85° C., from about 65° C. to about 80° C.,from about 65° C. to about 75° C., from about 65° C. to about 70° C., orfrom about 70° C. to about 95° C., or from about 70° C. to about 90° C.,from about 70° C. to about 85° C., from about 70° C. to about 80° C.,from about 70° C. to about 75° C., from about 75° C. to about 95° C.,from about 75° C. to about 90° C., from about 75° C. to about 85° C., orfrom about 75° C. to about 80° C., or from about 80° C. to about 95° C.,from about 80° C. to about 90° C., from about 80° C. to about 85° C., orfrom about 85° C. to about 95° C., from about 85° C. to about 90° C., orfrom about 90° C. to about 95° C. By comparison, in the previously knownconditions target specific primer concentrations are 10 nM to 400 nM;annealing time is between 10 sec and 2 min; and low annealingtemperatures are recommended (see Henegariu et al. (1997) “MultiplexPCR: Critical Parameters and Step-by-Step Protocol” BioTechniques 23:504-511; J. Brownie et al. (1997), in “the elimination of primer-dimeraccumulation in PCR” Nucleic Acids Res. 25: 3235-3241; and K. E. Varleyet al. (2008) “Nested Patch PCR enables highly multiplexed mutationdiscovery in candidate Genes” Genome Res. 18:1844-1850; B. Frey et al.(2013) “Methods and amplification of target nucleic acids using amulti-primer approach” US Patent Application Publication US2013/00045894 A1).

In some embodiments, the concentrations of all specific primers aresubstantially the same. In some embodiments, the concentrations ofdifferent specific primers are different. In some embodiments, theconcentrations of a selected number of primers are prepared atpredetermined levels and targeted at predetermined section of samplesequences for control purposes and/or for any other desired purposes. Insome embodiments, the concentration of a common primer is substantiallyhigher than the concentration of corresponding specific primer. In someembodiments, the concentration of a common primer is at least 50 nM, 100nM, 200 nM, 500 nM, 1,000 nM, 2,000 nM, 5,000 nM, or higher. Theconcentration of the common primer may be from about from about 10 nM toabout 5000 nM or from about 10 nM to about 1000 nM or from about 10 nMto about 500 nM or from about 10 nM to about 250 nM or from about 10 nMto about 100M or from about 25 nm to about 5000 nM, or from about 25 nMto about 1000 nM or from about 25 nM to about 500 nM or from about 25 nMto about 250 nM or from about 25 nM to about 125 nM or from about 25 nMto about 100 nM or from about 25 nm to about 50 nM or from about 50 nMto about 5000 nM or from about 50 nM to about 1000 nM or from about 50nM to about 500 nM or from about 50 nM to about 250 nM or from about 50nM to about 100 nM. In some embodiments, the molar concentration ratioof a common primer to a corresponding specific primer may be at least10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000,500,000 or greater. The ratio of the molar concentration ratio of acommon primer to a corresponding specific primer may be between 10 to5,000,000, or from 10 to 500,000, or from 10 to 50,000 or from 10 to5,000 or from 10 to 500 or from 10 to 50, or from 50 to 5,000,000 orfrom 50 to 500,000 or from 50 to 50,000 or from 50 to 5,000 or from 50to 500, or from 500 to 5,000,000 or from 500 to 500,000 or from 500 to50,000 or from 500 to 5,000. In some embodiments, the concentration of aspecific primer is at most 50 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5nM, 0.2 nM, 0.1 nM, 0.05 nM, 0.02 nM, 0.01 nM, 0.001 nM, or lower.

In some embodiments, annealing temperatures of cycle 1 and cycle 2 areselected to balance 3′ to 5′ nuclease activity and polymerase activityof the polymerase used. In some embodiments, annealing temperatures ofcycle 1 and cycle 2 are selected so that combined result of nucleaseactivity and polymerase activity of the polymerase used does not causesubstantial degradation of specific primers during the annealing period.

In some embodiments of using omega primers and polymerases containing 3′to 5′ exonuclease activities, phase I annealing steps consist of twostages of stage 1 and stage 2. The stage 1 temperature is close to or atpolymerase extension temperature while the stage 2 temperature is lowerthan that of stage 1. Stage 1 has a longer time or duration than that ofstage 2. Each omega primer is designed such that 5p arm is of sufficientlength that at the stage 1 temperature the 5p arm sequence forms astable hybridization with the corresponding template sequence while 3parm is sufficiently short that at the stage 1 temperature the 3p armremains largely in free form (unbound). This condition is designed tominimize 3′ exonuclease digestion of the omega primer during the longstage 1 annealing time which is designed so that the template sequencehybridized with the primer at a high ratio. The length of the 3p arm isdesigned in such a way that at the stage 2 temperature the 3p armhybridizes to the template.

Reaction conditions for cycle 1 and cycle 2 extension steps and phase 2thermo cycles may be set using general PCR conditions as found in theproduct instructions of corresponding polymerase suppliers. In someembodiments, when pluralities of target sequences of high GC contentsare involved, extension times may be extended. In some embodiments, theextension time is at least 15 seconds, 30 seconds, 60 seconds, 90seconds, 120 seconds, or more. Exemplary reaction conditions areprovided in the Examples of this description.

In some embodiments, spike-in controls are added into the sample inwhich multiplex PCR is performed. In one exemplary embodiment, thespike-in controls are chemically synthesized nucleic acids of knownsequences. In another exemplary embodiment, the spike-in controls areobtained by extracted biological nucleic acids of known sequences. Inanother embodiment, the spike-in controls are mixed chemicallysynthesized nucleic acids and extracted biological nucleic acids ofknown sequences. The sequences of the spike-in controls may be selectedfrom double stranded nucleic acid sequences, single stranded nucleicacid sequences, sequences containing 3′ and 5′ ends matching to 3′ endsof common primers and being able to be amplified by the common primers,and sequences containing 3′ and 5′ ends that do not match to 3′ ends ofcommon primers but match to corresponding specific primers and can bereplicated by the specific primers. In some embodiments, spike-incontrols include a plurality of nucleic acid sequences of different GCcontents. In one embodiment, the GC contents vary from 15% to 85% orfrom 15% to 80% or from 15% to 75% or from 15% to 70% or from 15% to 65%or from 15% to 60% or from 20% to 85% or from 20% to 80% or from 20% to75% or from 20% to 70% or from 20% to 65% or from 20% to 60% or from 25%to 85% or from 15% to 80% or from 25% to 75% or from 25% to 70% or from25% to 65% or from 25% to 60%. The actual GC content range of thespike-in controls for specific applications can be decided by oneskilled in the art of specific applications. In some embodiments,quantitative analysis of amplification product from samples containingspike-in controls is used to optimize reaction conditions. In someembodiments, the quantitative analysis of amplification product fromsamples containing spike-in controls is used for quality control. Insome embodiments, the quantitative analysis of amplification productfrom samples containing spike-in controls is used to performquantitative normalization.

Primer Fabrication

In some embodiments, primers may be made by conventional chemicalsynthesis (L. J. McBride et al. (1983) “An investigation of severaldeoxynucleoside phosphoramidites useful for synthesizingdeoxyoligonucleotides” Tetrahedron Letters, 24:245 248). In some otherembodiments, primers are made by chemical synthesis on microarrays (X.Zhou et al. (2004) “Microfluidic PicoArray synthesis ofoligodeoxynucleotides and simultaneously assembling of multiple DNAsequences” Nucleic Acids Res. 32:5409-5417; X. Gao et al. “Method andapparatus for chemical and biochemical reactions using photo-generatedreagents”. U.S. Pat. No. 6,426,184; Gao, X., Zhang, H., Yu, P.,LeProust, E., Pellois, J. P. Xiang, Q., Zhou, X. “Linkers andco-coupling agents for optimization of oligonucleotide synthesis andpurification on solid supports”. U.S. Pat. No. 7,211,654, AU2002305061;Gao, X., Zhou, X., Cai, S.-Y, You, Q., Zhang, X. “Array oligomersynthesis and use” WO2004/039953). The synthesis on microarrays has theadvantage of low per sequence cost and is particularly advantageous inhigh multiplex PCR applications wherein at least 10, 50, 100, 500,1,000, 5,000, 10,000, 50,000, or 100,000 primer sequences are required.Microarray synthesized oligonucleotide mixtures under the product nameof OligoMix™ are commercial available from LC Sciences (Houston, Tex.).

In some embodiments, synthetic oligonucleotides are amplified beforebeing used as PCR primers. Amplification is preferred when chemicalsynthesis scale is low, such as the case of chemical synthesis onmicroarrays. FIG. 9 illustrates an exemplary embodiment of producingspecific primers by amplification. Primer precursor templates 900 arechemically synthesized using either the convention method of the abovemicroarray method. Each primer precursor template 900 is anoligonucleotide comprising a number of functional segments including 5pflank 901, 5p arm 902, loop 903, 3p arm 904, and 3p flank 905. At the 5′end of 3p flank segment 905 is a dA (deoxyadenosine) nucleotide. Forillustration purpose, the primer precursor template 900 shown in FIG. 9is designed for preparing omega primers. However, the template designcan be made for preparing regular primers as well by simply replacingthe section between 5p flank 901 and 3p flank 905 with regular primersegments (e.g. specific segment 341 and common segment 342 of FIG. 3).Two preparation primers, prep primer 1 (906) and prep primer 2 (907) areused for PCR amplification. The sequence of prep primer 1 (906) issubstantially the same as that of 5p flank 901. The sequence of prepprimer 2 (907) is substantially complementary to that of 3p flank 905.Additionally, the 3′ end of prep primer 2 (907) is a dU (deoxyuridine)nucleotide. Although only one primer precursor template 900 is drawn inFIG. 9, the amplification is intended for simultaneous amplification ofmultiple primer precursor templates 900 to produce the specific primersfor multiple target selection and library amplification. All individualprimer precursor templates 900 have the same pair of 5p flank and 3pflank segments but have a specific primer section in the middle. The usethe dU containing primer requires PCR polymerases to have the ability toread through uracils. Acceptable polymerases include but are not limitedto Hot Start Taq DNA Polymerase from NEB (Ipswich, Mass.) and PfuTurboCx Hotstart DNA Polymerase from Agilent (Santa Clara, Calif.). Measuresshould be taken to minimize sequence bias and to avoid PCR product crosshybridization during the PCR amplification of the mixed templates. Hotstart polymerases are preferred. Melting temperatures of prep primer 1906 as well as prep primer 2 907 are preferably 5° C. or more above thehighest extension temperature suggested by the polymerase manufacture sothat both PCR annealing and extension temperatures can be maximized. LowPCR cycle numbers are preferred. In some embodiments, the cycle numberis at most 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, or lower. At thecompletion of precursor amplification, double stranded precursor 910 isproduced.

The next step is to uncap the precursor, in which the dU in the minusstrand 919 of precursor 910 is digested in a UDG/EDA (uracil-DNAglycosylase/ethylene diamine) solution. Alternatively, dU may bedigested using USER™ (Uracil-Specific Excision Reagent) Enzyme from NEB(Ipswich, Mass.). In some embodiments, a purification process is appliedafter the digestion to remove the digestion enzymes and/or to changebuffer compositions. The purification methods include but are notlimited to normalization beads from Axygen Biosciences (Union City,Calif.), PCR purification columns from Qiagen (Valencia, Calif.), andgel cut purification. In the digested precursor 920, 5′ end of 3p arm924 of the minus strand 929 is uncapped. Although FIG. 9 depicts thefragment 927 as being hybridized to the plus strand 928 of precursor920, in some embodiments the fragment 927 is dissociated from the plusstrand 928 either during digestion or during purification.

The lower portion of FIG. 9 illustrates how to activate the digestedprecursors into specific primers and how to integrate the activationprocess with target selection and amplification processes. The threefunctionally distinct processes, primer activation, target selection,and amplification may be carried out in a single tube. An exemplarystarting reaction mixture comprises the digested precursors 930, prepprimer 1 (936), templates 941, common primer 1 (942), common primer 2(943), one or more DNA polymerases, and dNTP containing PCR buffer. Thedigested precursors 930 are the same as the digested precursors 920 andthe prep primer 1 (936) is the same as primer 1 906. Suitableconcentrations of the digested precursors 930 and the prep primer 1(936) are similar to that of specific primers and common primers whichhave been described in section “Reaction conditions”. Other componentsare used to carry out relay PCR in a similar fashion as described aboveand their suitable concentrations have been describe in section“Reaction conditions”. The reaction is carried out on a PCR machine. Inthe first cycle, prep primer 1 (936) anneals with minus strands 939 andthen is extended to form activated specific primers 940 that haveextendable 3p arms. Starting from the second cycle the reaction proceedsas a relay PCR process in the same fashions as describe in previoussections and as illustrated in FIGS. 3, 4, 5 and 6. One aspect of thismethod is the in situ production of active specific primers. Theadvantage of the method is the simplicity of the process and the minimumnumber of steps involved.

FIG. 10 illustrates another exemplary embodiment of producing specificprimers by amplification. A PCR template 1000 comprises spc primersegment 1002 for specific primer, 5p flank segment 1001, and 3p flank1003. At the 5′ end of 3p flank segment 1003 is a dA (deoxyadenosine)nucleotide. The spc primer segment 1002 can be the sequence of a regularspecific primer, an omega specific primer, or any nucleotide sequence.Two preparation primers, prep primer 1 (1004) and prep primer 2 (1005)are used for PCR amplification. The prep primer 1 (1004) comprises a 3′section of which the sequence is substantially the same as that of 5pflank 1001. The prep primer 2 (1005) comprises a 3′ section of which thesequence is substantially complementary to that of 3p flank 1003. Theprep primer 2 (1005) further comprises a dU (deoxyuridine) at its 3′terminal. In some embodiments, it is preferred to have one or more dUsincorporated in to mid-section of prep primer 2 (1005). In someembodiments, multiple PCR templates 1000 are included in the processwith each template comprising distinct spc primer segment 1002 butidentical 5p flank segment 1001 and identical 3p flank segment 1003.Suitable PCR conditions have been described above relating to theprocess of FIG. 9. At the completion of PCR amplification, doublestranded product 1010 is produced.

The next step is to remove dUs in the minus strand 1019 of the doublestranded PCR product 1010. In some embodiments, dUs are removed ordigested in a UDG (uracil-DNA glycosylase) solution to remove uracilebase and then in EDA (ethylene diamine) solution to scissor nucleotidebackbone. In some embodiments, dUs are digested using USER™(Uracil-Specific Excision Reagent) Enzyme from NEB (Ipswich, Mass.).Both digestion processes produce minus strand 1029 with preferredphosphorylated 5′ terminals. In some embodiments, a purification processis applied after the digestion to remove the digestion enzymes and/or tochange buffer compositions. The purification methods include but notlimited to normalization beads from Axygen Biosciences (Union City,Calif.), PCR purification columns from Qiagen (Valencia, Calif.), andgel cut purification. In some preferred embodiments, one or more dUshaving been incorporated into mid-section of prep primer 2 (1005),remaining fragments of prep primer 2 are too short to stay on plusstrand 1028 and a single stranded 3p flank 1023 is obtained.

The next step is to end polish product 1020 by removing the singlestranded 3p flank 1023. The requirements for the end polish process areto produce a blunt ended double stranded product 1030 with the 3′ end ofthe plus strand 1038 to be the same as the 3′ end of spc primer 1002 andto keep the phosphorylated 5′ terminal of minus strand 1039 intact. Inan exemplary embodiment, the end polish is performed using T4 DNApolymerase from NEB (Ipswich, Mass.). T4 DNA polymerase has a 3′ to 5′exonuclease activity and does not have a 5′ to 3′ exonuclease function.The plus strand 1038 in the end polish product 1030 has a hydroxyl 3′terminal and can be used as an active specific primer in relay PCR. Theefficiency of the specific primer can be improved by removing thecounter strand of the active primer. The final step is to remove theminus strand 1039. In some embodiments, the minus strand 1039 is removedenzymatically. In an exemplary embodiment, Lambda exonuclease from NEB(Ipswich, Mass.) is used to digest the minus strand 1039. The finalproduct for this fabrication process is single stranded specific primer1040.

FIG. 11 illustrates another embodiment of producing specific primers byamplification. A PCR template 1100 comprises spc primer segment 1102 forspecific primer, 5p flank segment 1101, and 3p flank 1103. In someembodiments, the 3p flank segment 1103 comprises one or more restrictionsites to be recognized by one or more restriction enzymes. The spcprimer segment 1102 can be the sequence of a regular specific primer, anomega specific primer, or any nucleotide sequence. Two preparationprimers, prep primer 1 (1104) and prep primer 2 (1105) are used for PCRamplification. The prep primer 1 (1104) comprises a 3′ section of whichthe sequence is substantially the same as that of 5p flank 1101. Theprep primer 2 (1105) comprises a 3′ section of which the sequence issubstantially complementary to that of 3p flank 1103. The prep primer 2(1105) further comprises one or more restriction sites. In someembodiments, multiple PCR templates 1100 are included in the processwith each template comprising distinct spc primer segment 1102 butidentical 5p flank segment 1101 and identical 3p flank segment 1103. Ascompared to the PCR reactions of FIG. 9 and FIG. 10, the PCR componentsof FIG. 11 do not require dU, therefore the PCR polymerases used in themethod do not require the ability to read through uracils. Suitablepolymerases have been described in above “Polymerase selection” section.Measures should be taken to minimize sequence bias and to avoid PCRproduct cross hybridization during the PCR amplification of the mixedtemplates. Hot start polymerases are preferred. Prep primer 1 (1104) aswell as prep primer 2 (1105) are preferably designed in such a way thatPCR annealing and extension temperatures can be used at the highestpossible levels. Later sections of this disclosure will provide detaileddescriptions on primer designs. Low PCR cycle numbers are preferred. Insome embodiments, the cycle number is at most 25, 24, 23, 22, 21, 20,19, 18, 17, 16, 15, or lower. At the completion of precursoramplification, double stranded product 1110 is produced.

The next step is restriction digestion. This step is to remove 3p flank1103 from plus strand 1118 of the double stranded PCR product 1110 so asto expose the 3′ end of spc primer segment. In some embodiments, one ormore restriction enzymes are used to carry out the digestion. In someembodiments, type IIS restriction enzymes (A. Pingoud et al. (2001)“Structure and function of type II restriction endonucleases” NucleicAcids Res. 29 3705-3727) are utilized. In some embodiments, restrictionenzymes are obtained from commercial suppliers, such as NEB (Ipswich,Mass.). Exemplary restriction enzymes include but are not limited toBspQI, SapI, BsaI-HF, BpuEI, BfuAI, AcuI, and BtsIl from NEB (Ipswich,Mass.). In some embodiments, one of the considerations in the design ofthe template 1100, prep primer 1, and prep primer 2 is to confine therecognition site sequence in 3p flank 1103 and prep primer 2 and avoidthe recognition site sequence in any other sections of the template 1100and in prep primer 1 1104. In preferred embodiments, in the digestionproduct 1120, 3′ end of the plus strand 1128 is the same as the 3′ endof spc primer 1112. In preferred embodiments, in the digestion product1120, 5′ terminal of minus strand 1129 is phosphorylated and 3′ terminalof plus strand 1128 is a hydroxyl group.

The plus strand 1128 of the digestion product 1120 has a hydroxyl 3′terminal and can be used as an active specific primer in relay PCR. Theefficiency of the specific primer can be improved by removing thecounter strand of the active primer. The final step is to remove theminus strand 1129. In some embodiments, the minus strand 1129 is removedenzymatically. In an exemplary embodiment, Lambda exonuclease from NEB(Ipswich, Mass.) is used to digest the minus strand 1129. The finalproduct for this fabrication process is single stranded specific primer1130.

Primer and Target Sequence Design

In certain embodiments the form of specific primers consists of twofunctionally different sections, as shown in FIG. 3. The 3′ section istarget specific segment and 5′ section is common segment. In someembodiments, the omega form of the specific primers consists of threefunctionally different sections as shown in FIGS. 4A and 4B. The 3p armis the first target specific segment; the loop section is a commonsegment; and 5p arm is the second target specific segment. In someembodiments, as shown in FIGS. 3, 4A, 4B, and 5, the common primersconsist of two sections. The 3′ section is a common segment and 5′section is a tail segment.

In some embodiments, one pair of common primers is used. A pair normallyconsists of common primer 1 and common primer 2 where common primers 1and 2 consist of common segments 1 and 2 and tail segments 1 and 2,respectively. In embodiments using a regular form of specific primers,each pair of specific primers should contain a first specific primerconsisting of the common segment 1 and a first target specific segmentand a second specific primer consisting of the common segment 2 and asecond target specific segment. In embodiments using the omega form ofthe specific primers, each pair of specific primers should contain afirst specific primer consisting of the common segment 1, a first 3parm, and a first 5p arm and a second specific primer consisting of thecommon segment 2, a second 3p arm, and a second 5p arm.

In some embodiments, two or more pairs of common primers may be used.Then, a corresponding set of pairs of specific primers are used witheach pair of the common primers having a corresponding set of specificprimers. The composition and relationship among each set of the commonprimers and specific primers are the same as described in the aboveparagraph.

In general, common segment sequences of specific primers and commonprimers are designed to exhibit the following characteristics: (i) theyshould have no substantial hybridization to any expected or suspectedsequences in the samples of interest; (ii) they should have nosubstantial hybridization to themselves or to each other; and (iii) theyshould have no stable secondary structure. Additionally, 3′ ends of thecommon primers should produce substantially stable hybridization. Thisis generally achieved by having an adequate GC contents at the 3′ ends.The length and GC contents of the common segments of the common primersshould be designed to have melting temperatures no less than the phase 2thermo cycle annealing temperature.

In some embodiments, the present invention is used to prepare samplesfor massive parallel sequencing applications. The tail segments of thecommon primers are designed to accommodate clonal emulsionamplification, clonal bridge amplification, and/or any other reactionsinvolved in sequencing template preparation processes (E. R. Mardis(2008) “Next-Generation DNA Sequencing Methods” Annu. Rev. Genomics Hum.Genet. 9:387-402). In some embodiments, the tail segments of the commonprimers contain DNA barcode.

In general, the sequence of the target specific segment of a regularform specific primer is substantially identical or complementary to aselected portion of a target sequence of interest. The sequence isselected to exhibit the following characteristics: (i) it should haveminimal hybridization to any other portions of expected or suspectedsequences in the samples of interest; (ii) it should have no substantialhybridization to other specific primers, to common primers, and toitself; (iii) 3′ end of the primer should produce substantially stablehybridization with template; (iv) it should be sufficiently long so asto form substantially stable hybridization with corresponding templateat corresponding annealing condition; and (v) the specific primer aswhole (including specific and common segments) should have no stablesecondary structure.

The principles of selecting the sequences of 3p and 5p arms of omegaspecific primers are similar to that for regular form specific primersdepicted in the above paragraph. Following additional considerations aredirected towards the formation of omega forms. (i) Loop should haveminimal hybridization to any portions of expected or suspected sequencesin the samples of interest. (ii) The lengths of 3p arm and 5p arm shouldbe sufficiently long so that simultaneous hybridizations of both 3p armand 5p arm to corresponding template are substantially stable atcorresponding annealing condition. These structures are formed byhybridization interactions between primer and template sequences andthey can be designed with theoretical calculations by those of skilledin the art (see J. SantaLucia Jr. et al. (2004) “The thermodynamics ofDNA structural motifs” Annu. Rev. Biophys. Biomol. Struct. 33:415-440).

In some embodiments, specific and/or common primers contain modifiednucleotides for performance improvement and/or specific applications. Inan exemplary embodiment, 3′ ends of the primers are made ofphosphorothioate modified nucleotides. In one aspect, thephosphorothioate modified nucleotides inhibit exonuclease degradation.In some embodiments, the number of phosphorothioate modified nucleotidesat 3′ is at least 1, 2, 3, or more.

Computation Methods

This disclosure describes a rigorous PCR primer design method. Oneaspect of the method is the use of rigorous thermodynamic calculationsto quantitatively predict primer performances including on-targetprimer/target binding coefficient, priming efficiency, off-targetextension probability, and primer-dimer formation probability. Thequantitative aspect of the disclosed method is a significant improvementover the state of art primer design methods and/or tools which arelargely qualitative methods using empirically formulated scores todecide primer selections (A. Untergasser et al. (2012) “Primer3-newcapabilities and interfaces” Nucleic Acids Res. 40:e115). Another aspectof the method is the design of variant tolerant primers to achieverobust performance on samples from general populations. FIG. 12 depictsa flowchart of the disclosed computation process. In the followingdescription, human genome related sequences are mostly used as examples.However, the disclosed method can be used to design primers for anyspecies including artificial target sequences. This section of thespecification involves bioinformatics and thermodynamics. The disclosedmethods can be utilized to design PCR primers by those who are skilledin the art.

Step 1 of the primer design process is to prepare primer bindingtemplate sequences. The input data are user defined target regionsincluding database version, chromosome number, start and end positionsof the target regions. The input data may also include user suppliedsequence variations in the target regions. In a preferred embodiment, auser provides sequence variations of individual specimens or patients.In some embodiments, when the sequence variations from individualspecimens are not available, a user may provide combined sequencevariations. In some embodiments, a user may choose an alternative targetsequence input format by directly providing individual target sequences.

Based on the input data of target information, primer binding templatesequences for specific primer design are extracted from referencesequence database. The region of each primer binding template sequenceexceeds the corresponding target region by extending both ends of thetarget region so that sufficient room is available to place the primersfor capturing the entire target region. In some embodiments, theextension length is at least 50, 75, 100, 125, 150, 200, 250, or more.In some embodiments, where specific primers are used for preparinglibraries for high-throughput sequencing use, the extension length isselected to be approximate to the read length of the sequencing run. Thereference sequence database can be chosen from various public andprivate sources. Exemplary reference databases include Genome ReferenceConsortium GRCh37 and GRCh38 for human genome, GRCm38 for mouse genome,and CRCz10 for zebra fish genome. In a preferred embodiment, referencesequence databases with repeating sequences soft-masked may be used. Thesoft-masking converts repeating sequences into lower case letters whilemaintaining the rest of sequences in upper case letters. The extractedreference sequence is shown in FIG. 13 as reference allele 1300.

In some embodiments, when primers are designed for large populations, itis preferred to include variant sequences in the primer binding templateregions whenever the variant data is available. Variant databases may beobtained either from public or private sources. A preferred type ofvariant database comprises variants of individual specimens. An evenmore preferred type of variant database comprises haplotype variants ofindividual specimens. In this regard, the 1000 Genomes Project hasproduced and released a haplotype variant database of human genome (G.McVean et al. (2012) “An integrated map of genetic variation from 1,092human genomes” Nature, 491, 56-69). Alternatively, when variant databaseof individual specimens is not available, a variant database of combinedspecimens can be used. In this regard, dbSNP build 138 available fromUCSC Genome Browser for human SNP database is a combined variantdatabase.

Variant sequences in the primer binding template region are extractedfrom the best available variant database. In an exemplary embodiment,the variant sequences in the regions of interest are extracted fromHuman 1000 Genome database using VCF tools. The extracted data compriseshaplotype data of more than 1,000 individuals. In some embodiments, theextracted data is compiled into a list of unique haplotypes with allelefrequencies above a predetermined threshold value. In some embodiments,the predetermined threshold value is at most 10%, 5%, 1%, 0.5%, orlower. In some embodiments, the compiled haplotype data is formattedinto variant alleles 1310, 1320, 1330, 1340, 1350, and 1360 that arealigned to the reference allele 1300 of the primer binding templates asshown in FIG. 13. These variant alleles are useful in the design ofspecific primer sequences that satisfies the requirement of certainpercentage of success in a given population. In another exemplaryembodiment, the variant sequences in the regions of interest areextracted from a dbSNP by direct reading from fasta files. The extracteddbSNP sequence 1370 is aligned to reference allele 1300. In someembodiments, when user supplied variant sequences are present, thevariant sequences are compiled in a similar fashion as described above.

Next, the reference primer binding template sequences are profiled. Thisis to identify any sequence features that will require specialconsiderations during the determination of start and end positions ofindividual amplicons. In some embodiments, the profiling includes theidentification of masked repeating sequences and homologous sections.The locations of the masked repeating sequences within individualreference primer binding template sequences are extracted and compiledin a data table for later use. The homologous sections within as well asacross individual reference primer binding template sequences areidentified by alignment. In some embodiments, the alignment is performedusing alignment tools such as BLAST from NIH. In some embodiments, thelength of the homologous sections to be identified is at least 50, 75,100, 150, 200, 250, 300, 400, 500, or more. In some embodiments, thelength of the homologous sections to be identified is at least theaverage length of the captured regions. In some embodiments, the averagelength of the captured regions is predetermined by the primer designerbased on specific application requirements. The locations of thehomologous sections within individual reference primer binding templatesequences are compiled in a target data table for later use.

Step 2 of the primer design process is to design primer sequences and tocalculate primer binding properties against reference as well as varianttarget alleles. Primer sequences are derived from rigorous thermodynamiccalculations under the guidance of a predetermined set of parameters. Insome embodiments, the sequences of binding sections of each specificprimer are determined by the sequence of corresponding reference allele1300. In some embodiments, the sequences of binding sections of somespecific primers are determined by the sequence of certain variantalleles (e.g. 1310, 1320, 1330, 1340, 1350, or 1360, FIG. 13) ofinterest. The disclosed method includes a number of principlecalculations that can be used individually or in combination to designregular primers, omega primers, any other forms of primers, and/orhybridization probes.

In an exemplary embodiment, a regular specific primer is designed. Asdescribed in previous sections relating to FIG. 3, a regular specificprimer comprises a specific segment 341 and a common segment 342. Thecommon segment is a given sequence in this exemplary embodiment. Belowis described how to design the sequence of the specific segment and howto evaluate its priming performance: 1) fix 3′ position of the primer ata predetermined location of the reference primer binding template; 2)determine the strand of the template; 3) determine a suitable length ofthe specific segment that would produce a sufficient fraction oftemplate being hybridized by the primer, under the predeterminedconditions of template concentration, primer concentration, buffer salt(including e.g. Na+ and Mg++) concentrations, and annealing temperature;4) starting with a predetermined minimum length, extract thecomplementary sequence of the template with the given strand, the givenstarting position, and the given length (in some embodiments, theminimum length is at most 20 nucleotides, 15 nucleotides, 10nucleotides, or less); 5) append the extracted sequence to the 3′ end ofthe common segment to form the initial trial sequence; 6) calculate thebinding free energy ΔG between the trial sequence and the templatesequence. In some embodiments, the binding free energy is calculatedusing nearest neighborhood method (see J. SantaLucia Jr. et al. (2004)“The thermodynamics of DNA structural motifs” Annu. Rev. Biophys.Biomol. Struct. 33:415-440). In some embodiments the binding free energyis calculated using a computation package such as UNAFold (N. Markhamand M. Zuker (2008) UNAFold: software for nucleic acid folding andhybridization. In Keith, J. M., editor, Bioinformatics, Volume II.Structure, Function and Applications, number 453 in Methods in MolecularBiology, chapter 1, pages 3-31. Humana Press, Totowa, N.J. ISBN978-1-60327-428-9.). From the free energy ΔG, template associationfraction f_(a) (Equation 7) is derived from hybridization equilibriumequation (Equations 1 and 5), mass balance equations (Equations 2, 3,and 4), and thermodynamic equilibrium constant equation (Equation 6).

$\begin{matrix}{{C_{t} + C_{p}}\overset{K_{a}}{\Leftrightarrow}C_{c}} & {{Equation}\mspace{14mu} 1} \\{C_{c} = {f_{a}C_{t_{0}}}} & {{Equation}\mspace{14mu} 2} \\{C_{t} = {\left( {1 - f_{a}} \right)C_{t_{0}}}} & {{Equation}\mspace{14mu} 3} \\{C_{p} = {C_{p_{0}} - {f_{a}C_{t_{0}}}}} & {{Equation}\mspace{14mu} 4} \\{K_{a} = {\frac{C_{c}}{C_{t}C_{p}} = \frac{f_{a}}{\left( {1 - f_{a}} \right)\left( {C_{p_{0}} - {f_{a}C_{t_{0}}}} \right)}}} & {{Equation}\mspace{14mu} 5} \\{K_{a} = ^{\frac{{- \Delta}\; G}{RT}}} & {{Equation}\mspace{14mu} 6} \\{{f_{a} \approx \frac{1}{{\frac{1}{C_{p_{0}}K_{a}} + 1}\;}} = \frac{1}{\frac{1}{C_{p_{0}}^{\frac{{- \Delta}\; G}{RT}}} + 1}} & {{Equation}\mspace{14mu} 7}\end{matrix}$

In the equations, C_(t), C_(p), and C_(c) are template, primer, andprimer-template complex concentrations, respectively, at equilibriumstate; C_(t0) and C_(p0) are the initial template and primerconcentrations, respectively; K_(a) is equilibrium constant; T isannealing temperature; and R is ideal gas constant. Equation 7 isderived from Equations 5 and 6 assuming that initial templateconcentration C_(t0) is significantly less than initial primerconcentration C_(p0). In some embodiments, one may choose to derive aprecise solution of f_(a) simply by solving the second order equation ofEquation 5.

The f_(a) value obtained from Equation 7 may be compared with apredetermined threshold template association fraction f_(a,thr). In someembodiments, f_(a,thr) is at least 0.90, 0.91, 0.92, 0.93, 0.94, 0.95,0.97, 0.98, 0.99, or more. In some embodiments, f_(a,thr) is at fromabout 0.90 to 0.99, 0.91 to 0.99, 0.92 to 0.99, 0.93 to 0.99, 0.94 to0.99, 0.95 to 0.99, 0.97 to 0.99, 0.98 to 0.99. If f_(a) is less thanf_(a,thr) the length of the specific segment may be increased by 1 or bya predetermined incremental number and the above calculations isrepeated until f_(a) is above f_(a,thr). The resulting sequence is acandidate primer subjected to further evaluation.

The disclosed method makes a significant improvement over state of artmethod and/tool for primer length determination. In the disclosed methodthe primer length is determined based on the calculated fraction oftemplate being hybridized by the primer at actual annealing or reactiontemperature and reaction compositions. Priming efficiency of the primeris proportional to the calculated quantity. In comparison, the state ofart primer design methods and tools determine the length of a primerbased on primer Tm, which is a nucleic acid quality property that isunrelated to actual reaction temperature. In a thermodynamic calculation(J. SantaLucia Jr. et al. (2004) “The thermodynamics of DNA structuralmotifs” Annu. Rev. Biophys. Biomol. Struct. 33:415-440), Tm relates toenthalpy and entropy. Tm does not have a monotonic relationship withfree energy, which relates to enthalpy, entropy, and temperature, and Tmdoes not give a prediction for template association fraction f_(a). Theempirical rules of using annealing temperature 5° C. below or 3° C.above primer Tm in the state of art primer design methods does notwarrant a sufficient or predictable primer-template binding.

Calculations to evaluate the priming performance of the candidate primerare then performed. In some embodiments, the evaluation includes primerbinding to variant alleles, primer 3′ end binding stability, and foldingimpact to primer binding. The hybridization between the reference allelederived candidate primer and a variant allele forms a duplex containingone or more non-Watson-Crick motifs or mismatches that usually lead tothe increase of binding free energy and therefore the decrease oftemplate association fraction (see Equation 7). In some embodiments,wherein individual variant alleles are available, binding free energiesΔG between the candidate primer and all individual available variantalleles are calculated using the above described methods and tools,which include the thermodynamic calculation of nucleic acid duplexcontaining mismatches. Then calculate the corresponding templateassociation fraction f_(a) values using Equation 7. Pick the lowestf_(a) value as the worst case scenario template association fractionf_(a,min) of the candidate primer. In some embodiments, where only acombined variant sequence (e.g. the one from dbSNP) is available, theworst case scenario template association fraction f_(a,min) iscalculated against the combined variant sequence using the same methodas that for individual variant alleles.

This disclosed method includes a consideration in thermodynamiccalculations relating to the effect of enzymes on nucleic acid binding.In common practices of performing PCR using commercially suppliedpolymerases, the primer annealing temperatures is normally set atmanufacture suggest levels. The annealing temperatures for mostpolymerases are suggested to be 5° C. below primer Tm. However, theannealing temperatures for a new class of polymerases, including Phusionpolymerase, and Q5 polymerase (both are offered by NEB, Ipswich, Mass.),are suggested to be 3° C. above primer Tm. This disclosed method takesthe effect of polymerase and associated proteins on binding into accountof thermodynamic calculations. In some embodiments, the effect ofpolymerase and associated proteins on binding is counted as anequivalent salt. In an exemplary embodiment, an additional 75 mM isadded to the salt concentration in thermodynamic calculation of freeenergy in a reaction mixture involving Phusion polymerase. In someembodiments, the equivalent salt concentration of polymerase andassociated proteins is derived by curve fitting of experimental PCRproduct yields measured under a matrix of conditions. The variables ofconditions include primer specific segment length and annealingtemperature. In some embodiments, the experimental PCR is relay PCR. Insome embodiments, the effect of polymerase on binding is measured usingUV melting curve method (J. SantaLucia (1998) “A unified view ofpolymer, dumbbell, and oligonucleotide DNA nearest-neighborthermodynamics” Proc. Natl. Acad. Sci., 95, 1460-1465).

In some embodiments, the calculation for primer 3′ end binding stabilityis carried out with the thermodynamic calculations of binding freeenergies between the candidate primer and reference as well as variantallele templates. In some embodiments, the primer-template binding freeenergy with primer 3′ end binding the template is calculated usingregular nearest neighbor thermodynamics. Name the free energy ΔG_(close)for primer 3′ end close or 3′ end binding to corresponding template.Then, calculate the free energy by sequentially removing nearestneighbors from stacking energy terms (see page 418-419 of J. SantaLuciaJr. et al. (2004) “The thermodynamics of DNA structural motifs” Annu.Rev. Biophys. Biomol. Struct. 33:415-440) for a predetermined number ofbases starting from 3′ end of the primer or until a minimum free energyis reached. In some embodiments, the predetermined number of bases is atleast 1, 2, 3, or more. Name the free energy ΔG_(open,i), for opening atbase i. Equation 8 shows the equilibrium reaction between the bindingstates of primer 3′ open at base i and primer 3′ closed. ΔΔG_(i) is thefree energy difference between the two binding states. A primer isreactive in an extension reaction only when its 3′ end binds tocorresponding template. The fraction of a primer-template complex havingprimer 3′ closed is shown in Equation 12, which is derived from bindingequilibrium equation (Equations 8 and 9), mass balance equation(Equations 10), and thermodynamic equilibrium constant equation(Equation 11).

$\begin{matrix}{C_{{open},i}\overset{{{\Delta \; \Delta \; G_{i}} = {{\Delta \; G_{close}} - {\Delta \; G_{{open},i}}}},K_{{close},i}}{}C_{close}} & {{Equation}\mspace{14mu} 8} \\{K_{{close},i} = \frac{C_{close}}{C_{{open},i}}} & {{Equation}\mspace{14mu} 9} \\{C_{c} = {{C_{close} + {\sum C_{{open},i}}} = {C_{close}\left( {1 + {\sum\frac{1}{K_{{close},i}}}} \right)}}} & {{Equation}\mspace{14mu} 10} \\{K_{{close},i} = ^{\frac{{- \Delta}\; \Delta \; G_{i}}{RT}}} & {{Equation}\mspace{14mu} 11} \\{f_{close} = {\frac{C_{close}}{C_{c}} = {\frac{1}{1 + {\sum\frac{1}{{close},i}}} = \frac{1}{1 + {\sum ^{\frac{\Delta \; \Delta \; G_{i}}{RT}}}}}}} & {{Equation}\mspace{14mu} 12}\end{matrix}$

In the equations, C_(open,i) and C_(closed) are the equilibriumconcentrations of primer-template complex with primer 3′ end open atbase i and with primer 3′ end close, respectively; C_(c) isprimer-template complex concentration expressed in Equation 2;K_(closed,i) is equilibrium constant of the binding states of primer 3′open at base i and primer 3′ close; T is annealing temperature; and R isideal gas constant.

In some embodiments, one or more polymerases involved have 3′ to 5′exonuclease activity and one or more mismatches at primer 3′ end may beremoved during annealing time. The mismatches can be present when thePCR sample contains one or more variant alleles. In some embodiments,where the polymerase used has 3′ to 5′ exonuclease activity, the primer3′ end binding stability is measured by the fraction of primer-templatecomplex having at most 1, 2, 3, or more nucleotides open at primer 3′end. The exact formulations for the calculation can be derived by thoseskilled in the art of thermodynamics and by following the teaching ofthis disclosure. The exact length of allowed opening can be determinedexperimentally for specific polymerases and specific annealingconditions including annealing time.

The disclosed method makes a significant improvement over state of artmethods and/tools for the prediction of primer 3′ end binding stability.The disclosed method provides a quantitative prediction for the fractionof primer 3′ end binding to its template at actual reaction condition.In comparison, the state of art primer design methods and tools provideempirical scores that penalize primer sequences involving long steams ofA and Tat 3′ end. In this aspect, the state of art methods arequalitative and do not provides a quantitative prediction for the primerperformance.

FIG. 14 illustrates how primer and template folding impactprimer-template binding. The figure shows three parallel reactionsincluding relaxed primer P 1401 turning to a folded structure P′ 1402,relaxed template T 1402 turning to a folded structure T′ 1412, andprimer P 1401 and template T 1402 hybridizing into primer-templatecomplex PT 1420. K_(pf), K_(bf) and K_(pt) are equilibrium constants offolding and hybridization reactions, respectively. At equilibrium, thethree reactions are expressed in Equations 13, 14, and 15. Mass balancesare expressed in Equations 16 and 17. Thermodynamic equilibriumconstants of the three reactions are expressed in Equations 18, 19, and20.

$\begin{matrix}{K_{pf} = \frac{C_{p^{\prime}}}{C_{p}}} & {{Equation}\mspace{14mu} 13} \\{K_{tf} = \frac{C_{t^{\prime}}}{C_{t}}} & {{Equation}\mspace{14mu} 14} \\{K_{p\; t} = \frac{C_{p\; t}}{C_{p}C_{t}}} & {{Equation}\mspace{14mu} 15} \\{C_{p_{0}} = {C_{p} + C_{p^{\prime}} + C_{p\; t}}} & {{Equation}\mspace{14mu} 16} \\{C_{t_{0}} = {C_{t} + C_{t^{\prime}} + C_{p\; t}}} & {{Equation}\mspace{14mu} 17} \\{K_{pf} = ^{\frac{{- \Delta}\; G_{pf}}{RT}}} & {{Equation}\mspace{14mu} 18} \\{K_{tf} = ^{\frac{{- \Delta}\; G_{f\; t}}{RT}}} & {{Equation}\mspace{14mu} 19} \\{K_{p\; t} = ^{\frac{{- \Delta}\; G_{p\; t}}{RT}}} & {{Equation}\mspace{14mu} 20}\end{matrix}$

In the equations, C_(p), C_(t), C_(p′) and C_(t′) are the equilibriumconcentrations of the primer and template in relaxed and folded states,respectively; C_(pt) is the equilibrium concentrations of thehybridization product; C_(p0) and C_(t0) are the starting concentrationsof primer and template, respectively; ΔG_(pf) is the folding free energyof primer within the specific segment; ΔG_(tf) is the folding freeenergy of template within the priming segment; T is annealingtemperature; and R is ideal gas constant. In some embodiments, thefolding free energies are calculated using nearest neighborhood method(see J. Santa Lucia Jr. et al. (2004) “The thermodynamics of DNAstructural motifs” Annu. Rev. Biophys. Biomol. Struct. 33:415-440). Insome embodiments the folding free energy is calculated using acomputation package such as UNAFold (N. Markham and M. Zuker (2008)UNAFold: software for nucleic acid folding and hybridization. In Keith,J. M., editor, Bioinformatics, Volume II. Structure, Function andApplications, number 453 in Methods in Molecular Biology, chapter 1,pages 3-31. Humana Press, Totowa, N.J. ISBN 978-1-60327-428-9.). In mostapplication cases, starting primer concentration is significantly higherthan starting template concentration and the hybridization productconcentration is always less than starting template concentration, asshown in Equation 20. Combine Equations 13 through 17 and applycondition Equation 21 we obtain f_(a,fold) of Equation 22 showing thefraction of the template being hybridized by the primer in the presenceof competing primer and template folding reactions.

$\begin{matrix}{C_{p_{0}}C_{t_{0}} > C_{p\; t}} & {{Equation}\mspace{14mu} 21} \\{f_{a,{fold}} = {\frac{C_{p\; t}}{C_{t_{0}}} \approx \frac{1}{\frac{\left( {K_{pf} + 1} \right)\left( {K_{tf} + 1} \right)}{K_{p\; t}C_{p_{0}}} + 1}}} & {{Equation}\mspace{14mu} 22}\end{matrix}$

The disclosed method makes a significant improvement over state of artmethods and/tools for the prediction of folding impact onprimer-template binding. The disclosed method provides a quantitativeprediction for the fraction of a template being hybridized by a primerin the presence of competing primer and template folding reactions. Incomparison, the state of art primer design methods and tools provideempirical scores that penalize primer sequences having stable folding(negative folding free energy). In this aspect, the state of art methodsare qualitative and do not provides a quantitative prediction for theprimer performance.

In some embodiments, the calculations of Equations 13 through 22 areperformed on reference as well as all available variant alleles. Pickthe lowest f_(a,fold) value as the worst case scenario templateassociation fraction of the candidate primer. The performance of aprimer is predicted by the worst case scenario priming efficiency whichis obtained by multiplying the lowest f_(a,fold) of Equation 22 with thelowest f_(close) of Equation 12. In some embodiments, where savingcomputation time is desired, Equation 22 is applied to referencealleles, Equation 7 and Equation 12 are applied to reference as well asvariant alleles. The performance of a primer is predicted by thecombination of f_(a,fold), the reference template associate fractionf_(a,ref), the worst case scenario template associate fractionf_(a,min), and the worst case scenario fraction of primer 3′ closef_(close,min), is estimated according to Equation 23. This completes thedesign and performance prediction of a regular primer.

$\begin{matrix}{f_{prm} = {\frac{f_{a,\min}}{f_{a,{ref}}}f_{{close},\min}f_{a,{fold}}}} & {{Equation}\mspace{14mu} 23}\end{matrix}$

Repeat the design and performance prediction for the next primer bymoving the 3′ position of the primer to another predetermined locationon the reference primer binding template until the process is completedfor all predetermined locations. In some embodiments, the predeterminedlocations are arranged in a tiling pattern. In some embodiments, thetiling increment is 1, 2, 3, 4, or more nucleotides on a referenceprimer binding template. In some embodiments, the tiling is formed onboth plus and minus strands of the reference primer binding template. Insome embodiments, a primer designed at a predetermined locationcomprises a common segment complementary to the common segment of apredetermined common primer (common primer 1 or common primer 2). Insome embodiments, two sets of primers are designed with each setcovering all the predetermined locations but one set comprising a commonsegment complementary to the common segment of common primer 1 and theother set comprising a common segment complementary to the commonsegment of common primer 2.

As described earlier, an omega primer comprises a 5p arm, a loop, and a3p arm. The loop sequences are substantially determined by the 3′sections of corresponding common primers and are provided as a part ofpredetermined parameters. In some embodiments, the 5p arm of the primerfunctions as an anchor to provide a stable binding to the templatesequence while 3p arm checks for specificity of the binding and bringsthe loop into the extension product. In an exemplary embodiment, theprimer design begins from 3p arm. Similar to the design of a regularprimer, the 3′ position of the primer is fixed at a predeterminedlocation of the reference primer binding template and the initial traillength of the 3p arm is set at a predetermined value. In someembodiments, the predetermined initial trail length is 5, 6, 7, 8, 9 ormore. For computation purpose, assume that an omega primer already bindsto its template through 5p arm. The proper length of the 3p arm that isjust enough to overcome the positive free energy of the loop and to bindto its template with a sufficient binding coefficient is the desiredoutcome. For convenience, binding of the 3p arm to the template islabeled as the close of the 3p arm. The equilibrium reaction of 3p armopen and closed is shown in Equation 24, which is a first orderreaction. Equation 25 shows the free energy of the primer-templatecomplex with 3p arm open. The terminal free energy ΔG_(terminal) relatesto dangle motifs at 5p arm 3′ end. ΔG_(5pArm,stack) is the nearestneighbor stacking free energy of 5p arm. Equation 26 shows the freeenergy of the primer-template complex with 3p arm close. ΔG_(loop) isthe loop free energy. ΔG_(3pArm,stack) is the nearest neighbor stackingfree energy of 3p arm. Equation 27 shows the fee energy differencebetween states of 3p arm close and 3p arm open. The free energycalculations for all motifs involved in Equations 25, 26, and 27 aredescribed in detail by SantaLucia (J. SantaLucia Jr. et al. (2004) “Thethermodynamics of DNA structural motifs” Annu. Rev. Biophys. Biomol.Struct. 33:415-440). Equation 28 expresses the equilibrium condition ofthe reaction shown by Equation 24. Equation 29 is a mass balancesequation. Equation 30 is the thermodynamic equilibrium constant of thereaction shown by Equations 24.

$\begin{matrix}{C_{{3{pArm}},{open}}\overset{{\Delta \; \Delta \; G} = {{\Delta \; G_{{3{pArm}},{close}}} - {\Delta \; G_{{3{pArm}},{open}}}}}{}C_{{3{pArm}},{close}}} & {{Equation}\mspace{14mu} 24} \\{{\Delta \; G_{{3{pArm}},{open}}} = {{\Delta \; G_{terminal}} + {\Delta \; G_{{5{pArm}},{stack}}}}} & {{Equation}\mspace{14mu} 25} \\{{\Delta \; G_{{3{pArm}},{close}}} = {{\Delta \; G_{loop}} + {\Delta \; G_{{3{pArm}},{stack}}} + {\Delta \; G_{{5{pArm}},{stack}}}}} & {{Equation}\mspace{14mu} 26} \\{{\Delta \; \Delta \; G} = {{{\Delta \; G_{{3{pArm}},{close}}} - {\Delta \; G_{{3{pArm}},{open}}}} = {{\Delta \; G_{loop}} + {\Delta \; G_{{3{pArm}},{stack}}} - {\Delta \; G_{terminal}}}}} & {{Equation}\mspace{14mu} 27} \\{K_{{3{pArm}},{close}} = \frac{C_{{3{pArm}},{close}}}{C_{{3{pArm}},{open}}}} & {{Equation}\mspace{14mu} 28} \\{C_{p\; t} = {C_{{3{pArm}},{open}} + C_{{3{pArm}},{close}}}} & {{Equation}\mspace{14mu} 29} \\{K_{{3{pArm}},{close}} = ^{\frac{{- \Delta}\; \Delta \; G}{RT}}} & {{Equation}\mspace{14mu} 30} \\{f_{{3{pArm}},{close}} = {\frac{C_{{3{pArm}},{close}}}{C_{p\; t}} = {\frac{1}{\frac{1}{K_{{3{pArm}},{close}}} + 1} = \frac{1}{^{\frac{\Delta \; \Delta \; G}{RT}} + 1}}}} & {{Equation}\mspace{14mu} 31}\end{matrix}$

In the equations, C_(pt), C_(3pArm,close), and C_(3pArm,open) are theconcentrations of primer-template complex, primer-template complex with3p arm close, and primer-template complex with 3p arm open; T isannealing temperature; and R is ideal gas constant. By combiningEquations 28, 29, and 30, Equation 31 is derived which shows thefraction of omega primer having 3p arm binding to the template.

The f_(3pArm,close) value obtained from Equation 31 is then comparedwith a predetermined threshold value f_(3pArm,close,thr). In someembodiments, f_(3pArm,close,thr) is at least 0.90, 0.91, 0.92, 0.93,0.94, 0.95, 0.97, 0.98, 0.99, or more. In some embodiments, f_(a,thr) isat from about 0.90 to 0.99, 0.91 to 0.99, 0.92 to 0.99, 0.93 to 0.99,0.94 to 0.99, 0.95 to 0.99, 0.97 to 0.99, 0.98 to 0.99. Iff_(3pArm,close) is less than f_(3pArm,close,thr) increase the 3p armlength by 1 or by a predetermined incremental number and repeat theabove calculations until f_(3pArm,close) is above f_(3pArm,close,thr).

Next, the length of 5p arm is determined. For demonstration purpose, abulge loop (shown in FIG. 1C as 122) is used in our omega primer. In theomega structure of the bulge loop the position of 3′ end of 5p arm isset immediately next to 5′ end of 3p arm. For computation purpose, the5p arm is treated as an isolated binding sequence. The length of the 5parm is derived in a similar to that for regular primer specific segment.The derived 5p arm meets the requirement of producing a predeterminedthreshold template association fraction. In some embodiments, thethreshold value is at least 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.97,0.98, 0.99, or more. In some embodiments, f_(a,thr) is f at from about0.90 to 0.99, 0.91 to 0.99, 0.92 to 0.99, 0.93 to 0.99, 0.94 to 0.99,0.95 to 0.99, 0.97 to 0.99, 0.98 to 0.99. By having derived thesequences of 5p arm and 3p arm plus the predetermined loop sequence, wehave a complete candidate omega primer sequence that subjects to furtherevaluation.

A significant aspect of this disclosure is the design of varianttolerant primers to achieve robust performance on samples from generalpopulations. In an exemplary embodiment, the design is applied to anomega primer. The 3p arm of the variant tolerant omega primer is derivedin the same way as described above. The 5p arm of the variant tolerantomega primer is derived by carrying out iterative calculations ofEquations 1 through 7 against variant alleles with incremental 5p armlengths until the threshold template association fraction requirement ismet. This forces the length of 5p arm to increase so as to maintain asufficient binding to the templates even when one or more variants arepresent in the priming region. In some embodiments, a predeterminedmaximum length is used in the computation to confine the 5p arm lengthwithin a limit. In some embodiments, the predetermined maximum length isat least 40, 50, 60 or more. In some embodiments, the variants includedin the calculations are limited to SNP variants. The frequency of SNPvariants in general populations is by far the highest among all types ofvariants. The SNP tolerant primer design can significantly expand theaccessible regions for primer placement in genome sequences. In someembodiments, the principle of variant tolerant primers is applied in thedesign of regular or any other types of primers or probes.

The predictions of omega primers performance are similar to thatdescribed above for regular primers. In some embodiments, the predictionof 3′ end binding stability of an omega primer is performed usingEquations 8 through 12. This produces the worst case scenario fractionof primer 3′ close f_(3pClose,min). In some embodiments, the predictionof the folding impact on 5p arm binding to template is performed usingEquations 13 through 22. This produces the template association fractionof binding to 5p arm in the presence of folding competitionf_(a,5pArm,fold). In some embodiments, the template association fractionof template binding with a whole omega primer f_(a,omega) is calculatedusing Equations 1 through 7. In some embodiments, the prediction offolding impact on template binding with a whole omega primerf_(a,omega,fold) is performed using Equations 13 through 22.

In some embodiments, additional considerations are given to theprediction of the folding impact on 3p arm binding. FIG. 15schematically illustrates competitive folding reactions with the 3p armbinding to template. In this exemplary illustration, an omega primer1501 having its 5p arm 1503 bound to template 1502 is in equilibriumwith 4 metastable states of template 1512 being folded, primer 1521being folded, both primer 1531 and template 1532 being folded, and thedesirable form of the 3p arm 1545 binding to template 1542. Thesereactions are expressed in 4 independent equilibrium equations ofEquations 32 through 35. Mass balance is expressed in Equation 36.Thermodynamic equilibrium constants are provided in Equations 37 through40.

$\begin{matrix}{K_{{fold},{tpl}} = \frac{C_{{fold},{tpl}}}{C_{open}}} & {{Equation}\mspace{14mu} 32} \\{K_{{fold},{prm}} = \frac{C_{{fold},{prm}}}{C_{open}}} & {{Equation}\mspace{14mu} 33} \\{K_{{fold},{both}} = \frac{C_{{fold},{both}}}{C_{open}}} & {{Equation}\mspace{14mu} 34} \\{K_{bind} = \frac{C_{bind}}{C_{open}}} & {{Equation}\mspace{14mu} 35} \\{C_{p\; t} = {C_{open} + C_{{fold},{tpl}} + C_{{fold},{prm}} + C_{{fold},{both}} + C_{bind}}} & {{Equation}\mspace{14mu} 36} \\{K_{{fold},{tpl}} = ^{\frac{{- \Delta}\; G_{{fold},{tpl}}}{RT}}} & {{Equation}\mspace{14mu} 37} \\{K_{{fold},{prm}} = ^{\frac{{- \Delta}\; G_{{fold},{prm}}}{RT}}} & {{Equation}\mspace{14mu} 38} \\{K_{{fold},{both}} = ^{\frac{- {({{\Delta \; G_{{fold},{tpl}}} + {\Delta \; G_{{fold},{prm}}}})}}{RT}}} & {{Equation}\mspace{14mu} 39} \\{K_{bind} = ^{\frac{{- \Delta}\; \Delta \; G_{3{pArmClose}}}{RT}}} & {{Equation}\mspace{14mu} 40} \\{f_{{3{pArmClose}},{fold}} = {\frac{C_{bind}}{C_{pt}} = \frac{K_{bind}}{1 + K_{bind} + K_{{fold},{tpl}} + K_{{fold},{prm}} + K_{{fold},{both}}}}} & {{Equation}\mspace{14mu} 41}\end{matrix}$

In the equations, C_(pt), C_(open), C_(fold,tpl), C_(fold,prm),C_(fold,both), and C_(bind) are the concentrations of primer-templatecomplex of all states with the 5p arm binding to template,primer-template complex of open state with primer and template inunfolded states, primer-template complex with template being folded,primer-template complex with primer being folded, primer-templatecomplex with both template and primer being folded, primer-templatecomplex with 3p arm binding to template; ΔΔG_(3pArmClose) is the freeenergy difference between the states of 3p arm closed and 3p arm openwhile 5p arm remains bound to template which value is provided inEquation 27; T is annealing temperature; and R is ideal gas constant.Combining Equations 32 through 36, derives Equation 41 which shows thefraction of omega primer having 3p arm binding to the template in thepresence of competing primer and template folding reactions.

In an exemplary embodiment, the performance of an omega primer ispredicted by the combination of the template associate fraction of theworst case scenario variant template binding with 5p armf_(a,5pArm,min), the template associate fraction of reference templatebinding with 5p arm f_(a,5pArm,ref), the worst case scenario fraction ofprimer 3′ close f_(3pClose,min), the template association fraction ofreference allele template binding to 5p arm in the presence of foldingcompetition f_(a,5pArm,fold), and the fraction of omega primer having 3parm binding to the template in the presence of competing primer andtemplate folding reactions f_(3pArmClose,fold). Equation 42 shows anexemplary performance prediction for an omega primer.

$\begin{matrix}{f_{prm} = {\frac{f_{a,{5{pArm}},\min}}{f_{a,{5{pArm}},{ref}}}f_{{3{pClose}},\min}f_{a,{5{pArm}},{fold}}f_{{3{pArmClose}},{fold}}}} & {{Equation}\mspace{14mu} 42}\end{matrix}$

In some embodiments, the template association fractions relating to 5parm binding (f_(a,5pArm,min), f_(a,5pArm,ref), and f_(a,5pArm,fold)) canbe replaced by the corresponding template association fractions relatingto whole omega primer binding, depending on the considerations ofcomputation time, calculation precision, specific designed functions of5p arm, 3p arm, and loop segments, and specific applications. Thecalculations of the template association fractions relating to wholeomega primer binding have been described above.

The design and performance prediction for the next omega primer may berepeated by moving the 3′ position of the primer to anotherpredetermined location on the reference primer binding template untilthe process is completed for all predetermined locations in fashion asdescribe above for regular primers.

In some embodiments, wherein the fabrication of the specific primersinvolves PCR amplification, the prediction of the primer performanceincludes the amplification efficiencies of PCR templates 900, 1000 and1100 of the primers (regular, omega, and any other types). In someembodiments, template folding in template flanking segments 901, 905,1001, 1003, 1101, and 1103 are calculated for the impact to primingefficiencies in PCR reactions. In some embodiments, the calculations arecarried out using Equations 13 through 22.

Steps 3 to 5 of the primer design process searches for primer off-targetlocations and provides quantitative predictions for the primer extensionefficiencies at the off-target locations. In step 3, all candidateprimers are aligned to a reference genome database or any sequencedatabase that substantially represents a complete DNA sequence setinvolved in the samples on which the candidate primers are going to beapplied. In some embodiments, the alignment is carried out by usingBLAST or one or more of BLAST derivatives. For convenience, thealignment results are labeled as hits. Remove the one-target hits fromthe total hits. An on-target hit is the sequence site that a primer isdesigned to bind. The remaining hits are the off-target hits. Save thealignment locations, aligned part of query sequence, and aligned part ofsubject sequence for off-target binding coefficient calculation uses.Primer sequences are the query sequences of the alignment and databasesequences are the subject sequences. In some embodiments, BLASTN usused. In some embodiments, BLAST is set up in such a way that mismatchesand gaps are allowed. In some embodiments, default values of BLASTgapopen, gapextend, penalty, and award are used. In some embodiments,BLAST is set up in such a way that mismatches and gaps are allowed. Insome embodiments, a reasonably small BLAST word_size is used to ensurethe sensitivity of the off-target search. In some embodiments, BLASTword_size is set at 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more. Theselection of the word_size is based on the balance of computation time,available computer memory, and the desired sensitivity of the alignmentsearch. BLAST is the most popular and basic alignment tool and its useis familiar by those skilled in the art of bioinformatics.

In some embodiments, wherein an off-target search is performed for omegaprimers, added alignment operations are desired in addition to thealignment of original whole omega primer sequence. The added alignmentcaptures any off-target sites that may hybridize with the omega primerand form stable loop structures. Being a local alignment tool, BLAST maymiss this type of sites due to large gaps involved. In some embodiments,the added alignment is formed by aligning the combined 5p arm and 3p armsequences against the sequence database of interest. In the alignmentresult when the aligned part of query sequence includes the junction of5p arm and 3p arm, insert the corresponding loop sequence into thejunction of the aligned part of query sequence to produce a restoredquery sequence and insert a gap of the same loop length into thejunction corresponding site of the aligned part of subject sequence toproduce an aligned subject sequence. Then compile the alignment resultsby removing redundant alignment hits between the results of whole omegaprimer alignment and combined 5p arm and 3p arm sequence alignment. Insome embodiments, the added alignment is performed by doing pairwisealignments using a global alignment tool on the sequence pairs of wholeomega primer sequences and expanded regions of corresponding BLAST hits.In some embodiments, the global alignment tool is written based onNeedleman-Wunsch algorithm (S. Needleman el al. (1970) “A general methodapplicable to the search for similarities in the amino acid sequence oftwo proteins” Journal of Molecular Biology 48, 443-53). The alignmentresult would already have the proper gaps.

Step 4 of the primer design is to construct primer/off-target bindingpair sequences ready for the binding coefficient calculation. The pairedsequences are constructed by expanding the aligned part of query as wellas subject sequences at both ends by a predetermined number ofnucleotides. In some embodiments, the predetermined number is 0, 1, 2, 3or more. The purpose of expanding the aligned parts is to produceoverhangs at both ends of the binding pair sequences so that thefollowing binding calculations can be done more accurately by includingthe overhang stacking energies (J. SantaLucia Jr. et al. (2004) “Thethermodynamics of DNA structural motifs” Annu. Rev. Biophys. Biomol.Struct. 33:415-440).

Step 5 of the primer design is to calculate the primer/off-targetbinding properties. The binding free energies between primers andoff-target sequences are calculated using the same methods describedabove for primer binding to on-target template. From the free energieswe obtain the binding coefficient on off-target sequences usingEquations 1 through 7 at specific primer annealing condition. Anotherimportant off-target binding property is the distance between the end ofthe binding regions and 3′ end of the primers also known as the distanceoverhang length. When the overhang length is below a threshold length,the off-target primer is considered extendable. In some embodiments, thethreshold length is determined by the polymerase used. In someembodiments, when the polymerase does not have 3′ to 5′ exonucleaseactivity, the threshold length is 0. In some embodiments, when thepolymerase has 3′ to 5′ exonuclease activity, the threshold length is atleast 0, 1, 2, 3 nucleotides, or more. In some embodiments, the full setof primer/off-target binding properties, including location, bindingcoefficient, and extendibility, are saved for further use when thebinding coefficient exceeds a threshold binding coefficient value. Insome embodiments, the threshold binding coefficient value for anextendable off-target binding is 0.05, 0.01, 0.005, or less. In someembodiments, the threshold binding coefficient value for anon-extendable off-target binding is 0.75, 0.5, 0.25, 0.1 or less. Thethreshold binding coefficient values are determined by the requiredprimer specificity, computation time, computer memory size, and computerstorage size.

Step 6 of the primer design is to calculate cross-hybridization bindingbetween candidate specific primers and common primers. In someembodiments, the common primers are aligned against all candidateprimers. Then the alignment results are used to calculate bindingcoefficients in the same way as described in Step 5. In someembodiments, the cross-hybridization free energies are calculated usinga computation package such as UNAFold (N. Markham and M. Zuker (2008)UNAFold: software for nucleic acid folding and hybridization. In Keith,J. M., editor, Bioinformatics, Volume II. Structure, Function andApplications, number 453 in Methods in Molecular Biology, chapter 1,pages 3-31. Humana Press, Totowa, N.J. ISBN 978-1-60327-428-9). For mostapplications, the concentration of each specific primer is significantlyless than that of the common primer and the binding coefficient is thefraction of a specific primer binding hybridized by a common primer. Theprimer extendibility for both specific primers and for common primersshould be examined. If an extendible primer is found, further examine ifthe extension product can be amplified by one or both common primers. Anextension product is amplifiable if it is produced by a common primerextending from the specific segment of a regular primer or from 3p armsegment of an omega primer. Record the binding coefficient,extendibility, and amplifiability of a candidate primer when the bindingcoefficient exceeds a threshold binding coefficient value. In someembodiments, the threshold binding coefficient value for anon-extendable cross-hybridization binding is 0.25, 0.2, 0.1 or less. Insome embodiments, the threshold binding coefficient value for anextendable but not amplifiable cross-hybridization binding is 0.2, 0.1,0.05, or less. In some embodiments, the threshold binding coefficientvalue for an extendable and amplifiable cross-hybridization binding is10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, or less. The threshold binding coefficientvalues for non-amplifiable cross-hybridization binding are determined bythe considerations of keeping a reasonably high fraction of the specificprimer available for specific priming use. The threshold bindingcoefficient value for amplifiable cross-hybridization binding isdetermined by the concentration ratio between specific primer and relayPCR template. The objective is to keep the level of primer-dimer productbelow the level of template amplification product. As an illustrativeexample, assuming the template concentration is 1 fM and the specificprimer concentration is 10 pM; a binding coefficient of 10⁻⁵ for anamplifiable cross-hybridization binding would produce about 0.1 fM ofprimer-primer extension product that would be amplified in parallel withthe template. At end, about 10% of the PCR product would beprimer-primer dimer. The calculation on the cross-hybridization bindingamong specific primers is performed in Step 9 of the computation processwhen individual candidate specific primers are actually picked.

Step 7 of the primer design is to calculate the scores of all candidateprimers. In some embodiments, in addition to the quantitativelycalculated priming efficiency derived in Step 2, we use several qualityscores to guide the final primer selection from the candidate primerpool. In an exemplary embodiment, the quality scores are formulated tofavor GC content of the primer between 0.4 and 0.6, to discourage primerlength beyond 90, to favor high ratio of primer lengths obtained onreference allele versus on the worst case scenario variant allele, todiscourage off-target binding, and to discourage cross-hybridizationbinding between specific primers and common primers. The calculations ofthe previous steps have already generated the required parameters neededfor the score calculations. The already calculated parameters includethe primer length, primer length against reference allele template, theprimer length against worst case scenario variant allele template, thebinding coefficients for the extendable and the non-extendableoff-target binding, and binding coefficients for the non-extendable,extendable but not amplifiable, and amplifiable cross-hybridizationbinding between candidate specific primers and common primers. Scoreformulations using linear, non-linear, proportional,reverse-proportional, and certain types of distribution curves which arefamiliar to those skilled in the art of process control, automation, andconventional PCR primer design. In an exemplary embodiment, all scoreshave a maximum value of 1 and minimum value of 0, 1 being the best and 0being the worst. Combine all individual scores into a single scoreaccording to Equation 43 to represent the overall quality of the primer.

S_(prm) =ΠS _(i) ^(w) ^(i)   Equation 43

In the equation, S_(i) is an individual score, including that of GCcontent, primer length, primer length ratio of reference allele versusthe worst case scenario variant allele, off-target binding, andcross-hybridization binding between specific primers and common primers.In the equation, w_(i) is the weighing factor of score S_(i). Theweighing factor is an empirically determined number that is used toadjust the relative importance of an individual score in thedetermination of overall quality score. The weighing factor usually hasa minimum value of 0.

Step 8 of the primer design is to build a list of candidate captureregions. The list comprises start and end locations of the captureregions with each candidate capture region having specific primer 1 andspecific primer 2. The candidate capture regions serve the purpose ofproviding full coverage of the requested target regions with abundantcombinations of specific primer 1 and specific primer 2 so that finalselections of the capture regions can be made by picking the bestpossible combinations of specific primer 1 and specific primer 2.“Candidate capture region” and “capture region” are usedinterchangeably. In some embodiments, the capture regions are arrangedin a tiling pattern. In some embodiments, the lengths of the captureregions are predetermined. In some embodiments, the predetermined regionlengths are decided based on applications. In an exemplary embodiment,the captured sequences are used in high-throughput sequencing. Thelengths of the capture regions are determined based on sequencing readlength. The sequencing read length is the number of nucleotides that asequencing instrument reports from a single reading pass. As anillustrative example, a sequencing run includes two passes with eachpass having a read length of 150 nt (nucleotide). The two passes readtarget sequences from two opposite ends. The total sequenced lengthwould be 300 nt. Then, for the purpose of reading the whole captureregion and yet having some overlapping between two reading passes, 280nucleotide can be selected as the maximum capture region length. It isuseful to maintain some flexibility on the region length for choosingthe best possible specific primers. 140 nucleotides was selected as theminimum capture region length. The ability to handle situations of along repeat masked section exceeding the maximum capture region lengthis also useful. A repeat masked sequence can often have a very largenumber of occurrences in a genome and is often masked out from primerdesign. In order to fully cover all requested target regions, themaximum capture region length is expanded when the tiling process runsinto a repeat masked region. For illustrative purpose, we set theexpanded maximum capture region length at 600.

In some embodiments, the tiling process for a target starts by pickingup the left most (or the lowest location) candidate specific primer 1 ofplus strand and then pick a candidate specific primer 2 of minus strandlocated a minimum capture region length upstream. Make sure at least aportion of requested target region is between the two specific primers.A candidate amplicon will be produced from PCR amplification of thecandidate captured sequence. The quality score of the candidate ampliconis the product of the quality scores of the two specific primers. Insome embodiments, other quality scores are calculated including but notlimited to GC content, folding free energy, and the length of thecandidate capture region. The formulation of the scores may be derivedby following the teaching of Step 7 above. The overall quality score ofthe amplicon is calculated the same as Equation 43. Add the start andend locations of the capture regions, the information of specific primer1, the information of specific primer 2 and the amplicon score to thelist of the candidate capture regions. Repeat the above process with anincrement of capture region length until the maximum capture regionlength is reached. In some embodiments, the increment is 1, 2, 3, 4, ormore. Then start from the next left most candidate specific primer 1 ofplus strand and repeat the above process until the tiling reaches to theright most available candidate specific primer. In some embodiment,additional rounds of tiling are performed by starting with the left mostcandidate specific primer 2 of plus strand, specific primer 1 of minusstrand, and specific primer 2 of minus strand, each matched by the otherspecific primer of the opposite strand.

Step 9 of the primer design is to select amplicon sequences to cover allrequested target sequences. In this section of the computation, finalselections of amplicons including corresponding specific primers aremade among all available candidate amplicons on the list of thecandidate capture regions. In some embodiments, final amplicons areselected in a tiling fashion. In some embodiments, the tiling process isapplied to one target sequence at a time.

In some embodiment, the tiling selection for a target sequence startsfrom the group of candidate amplicons that cover the starting positionof the target sequence. Among the group, calculate cross-hybridizationbinding between the paired candidate specific primers and each candidatespecific primer to itself in the same way as described in Step 6 above.Calculate the scores for the cross-hybridization binding in the same wayas described in Step 7 above.

Next, predict the capture of off-target sequences by the candidatespecific primers. This is done by looking up at the primer/off-targetbinding property data of Step 5 and deciding if any measurableoff-target sequences can be captured by the candidate specific primers.When two specific primers both have extendable off-target binding siteson the same contig sequence, have 3′ ends facing each other, are locatedin opposite strands, and are sufficiently close to each other, theywould produce an off-target sequence. The considerations include asingle specific primer having two or more off-target binding sites. Insome embodiments, the off-target capture is considered only when thedistance between the off-target binding sites is below a thresholddistance. In some embodiments, the threshold distance is at most100,000, 10,000, 1000, or less. The relative concentration of theoff-target product is predicted from the product of the bindingcoefficients of the two off-target binding sites. An off-target scorefor the candidate amplicon (which is associated with the candidatespecific primers) is calculated based on the relative concentration ofthe off-target product. In some embodiments, the off-target score isformulated as inversely proportional to the relative concentration ofthe off-target product, with the score ranging from 0 to 1.

Revise the quality score of the corresponding amplicon by multiplyingthe original quality score by the cross-hybridization binding score andthe off-target score. Then, pick the amplicon that has the highestquality score as the first tile. If the selected amplicon covers the endposition of the target sequence, the selection is complete for thetarget sequence. If the selected amplicon does not cover the endposition of the target sequence, proceed to the next tile.

In a preferred embodiment, adjacent tiles overlap for certain length atthe junction. In some embodiments, the overlap length is at least 1, 2,5, 10, 15 or more nucleotides. In some embodiments, a minimum overlaplength and a maximum overlap length are predefined as a part of inputparameters. In some embodiments, the minimum overlap length is at least1, 2, 5 or more nucleotides. In some embodiments, the maximum overlaplength is at least 10, 20, 30, or more nucleotides. In some embodiments,in order to avoid the interference of capture reactions (Cycle 1 andCycle 2 of FIGS. 3, 4A, 4B, and 5) between adjacent tiles, relay PCRreactions of adjacent tiles are performed in separate PCR tubes. In someembodiments, two PCR tubes are used for the capture and amplification ofa complete set of target sequences with each tube containing thespecific primers for every other member of the tiles. Save the primerinformation of the first tile amplicon to tube 1 primer list. The secondtile is selected from the group of candidate amplicons that have captureregion start position in the range of the first tile capture region endposition minus the maximum overlap length and minus the minimum overlaplength. From the group, pick the second tile amplicon in the same way asdescribe above for the first tile. Save the primer information of thesecond tile amplicon to tube 2 primer list. If the selected ampliconcovers the end position of the target sequence, the selection iscomplete for the target sequence. If the selected amplicon does notcover the end position of the target sequence, proceed to the next tileand until the end of the target sequence is reached.

When primers are already present in tube 1 or 2, the pre-existingprimers need to be included in the calculation of specific primercross-hybridization binding and off-target capture. For example, if theabove process is continued for the third tile, the newly selectedprimers would be placed in tube 1. The calculations for both specificprimer cross-hybridization binding and for off-target capture wouldinclude the binding between the paired new specific primers, each newspecific primer to itself, and each new specific primer to each one ofthe primers already in tube 1.

In some embodiments, special considerations are given to tile selectionson target sequences containing long homologous sections. As describedabove, the long homologous sections have been identified in Step 1 andthe information is available in a target data table. In someembodiments, amplicons are selected based on one copy of the homologoussections and the corresponding specific primers are used for all thehomologous sections. This avoids the potential problems of duplicatingand/or conflicting primer selections from essentially the same targetsequences but at different locations.

Obviously many modifications and variations of the disclosed computationmethod are possible in the light of the above teachings. For example, insome embodiments, low quality primers are removed along the way so as toreduce computation times for later steps.

Applications

In addition to the aforementioned target enrichment for massive parallelsequencing use, the present invention may be advantageously used invarious other applications. In some embodiments, omega primers incombination with Relay PCR are advantageously used in real-time PCR. Apair of specific primers and a pair of common primers are used in eachreaction. During a thermal cycling process, in the first two cyclesspecific Omega primers pick up a target section with high specificityand efficiency. In the remaining cycles common primers take over for theamplification of the target. The advantage of this approach is that onepair of well characterized common primers are used in amplificationcycles no matter what target sequences are. Measurement dependence overtarget sequence variation is expected to be reduced over conventionalreal-time PCR in which specific primers are responsible for allamplification cycles. Amplification yield differences due to differentspecific primer designs are exponentially amplified in the conventionalmethod. By using relay PCR, amplification yield differences due todifferent specific primer designs are eliminated. In some embodiments,regular specific primers in combination with Relay PCR are used inreal-time PCR.

In some embodiments, relay PCR is used to prepare samples for arraydetection uses. A plurality of specific primers and a pair of commonprimers are used in each relay PCR reaction. In some embodiments, atleast one common primer is attached with a fluorescence dye, includingbut not limited to Cy3, Cy5, Alexa 3, Alexa 5, FTIC, and FAM (add morehere). In some embodiments, at least one common primer is attached witha conjugation ligand, including but not limited to biotin, NHS, NH2, andCHO. The fluorescence dyes and the ligands are hereafter called labels.One or more labels may be attached to one or more nucleotides of aprimer molecule. A label attached primer is called labeled primer. Insome embodiments, a pair of common primers consists of one unlabeledprimer and one labeled primer. In some embodiments, the concentration ofthe labeled primer is higher than that of the unlabeled primer. Themolar concentration ratio of the labeled and unlabeled primers is atleast 1, 2, 5, 10, or greater. In some embodiments, two rounds of PCRreactions are used to produce labeled target samples. In the firstround, a relay PCR is performed. A plurality of specific primers and onepair of unlabeled common primers are used. The first round PCR productis optionally purified to remove residual primers and enzymes and toretain the double stranded PCR product. The second round is asingle-strand PCR reaction, involving one labeled common primer and analiquot of first round PCR product or purified first round PCR product.In some embodiments, the aliquot is at least 1/1000, 1/500, 1/200,1/100, 1/50, 1/20, 1/10 or more of the total volume of the first roundPCR product.

In some embodiments, the composition includes one or more targetspecific primer pairs that can amplify a short tandem repeat, singlenucleotide polymorphism, gene, exon, coding region, exome, or portionthereof. In some embodiments, templates are cDNAs that are synthesizedfrom RNA samples.

Thus, in some embodiments, an oligonucleotide primer comprising a 3p armhaving a 3′ end and a 5′ end, a loop section and a 5p arm having a 3′end and a 5′ end, wherein the 5p arm hybridizes to a DNA template andwherein the 3p arm hybridizes to the DNA template and provides sequencespecificity for polymerase extension and wherein the loop section islocated between the 5p arm and the 3p arm and does not bind the DNAtemplate is provided. In some embodiments, the DNA template issubstantially complementary to the 5p arm and the 3p arm. In someembodiments, the 5p arm is from 10 to 100 nucleotides in length, such as25 to 60 nucleotides, and/or the 3p arm is from 6 to 60 nucleotides inlength, such as from ten to 20 nucleotides, and/or the loop section isfrom 12 to 50 nucleotides in length, such as from 15 to 40 nucleotides.In some embodiments, the 5′ end of the 3p arm and the 3′ end of the 5parm are adjacent each other when bound to the DNA template. In someembodiments, the 5p arm has higher binding energy than the 3p arm whenhybridized to the DNA template (such as two or three times higher). Insome embodiments, the primer comprises a bulge loop, hairpin loop and/orinternal loop.

In some embodiments, this disclosure provides a hybridization structurefor assay use comprising a probe and a target wherein the hybridizationstructure has one or more single stranded loops and two or more duplexsegments wherein each loop is located between the duplex segments. Insome embodiments, the single stranded loop is in the probe and comprisesone or more non-nucleotide moieties. In some embodiments, the probecomprises a spacer. In some embodiments, the hybridization structure isused for polymerase extension. In some embodiments, the hybridizationstructure is used for hybridization detection. In some embodiments, theloop contains 12 to 50 nucleotides.

In some embodiments, methods for amplifying a target nucleic acid areprovided. In some embodiments, the method comprises providing a firstspecific primer, a first common primer, a second common primer, aflanked target fragment, a polymerase and nucleotides; performing atarget selection comprising one cycle of a first thermocycling routinecomprising an denaturation step, annealing step and an extension step;and, performing amplification comprising two or more cycles of a secondthermocycling routine comprising an denaturation step, annealing stepand an extension step thereby amplifying the target nucleic acid. Insome embodiments, the method comprises providing a first specificprimer, a second specific primer, a first common primer, a second commonprimer, a target nucleic acid, a polymerase and nucleotides; performinga target selection comprising two cycles of a first thermocyclingroutine comprising an denaturation step, annealing step and an extensionstep; and, performing amplification comprising two or more cycles of asecond thermocycling routine comprising an denaturation step, annealingstep and an extension step thereby amplifying the target nucleic acid.In some embodiments of these methods, the first specific primer has a 3′end and a 5′ end wherein the 3′ end contains a first sequence specificsegment and the 5′ end contains a first common segment, and/or thesecond specific primer has a 3′ end and a 5′ end wherein the 3′ endcontains a second sequence specific segment and the 5′ end contains asecond common segment, and/or the concentration of the first and secondspecific primers is 500 fold less than that of the first and secondcommon primer, and/or the concentration of the first and second specificprimers is from about 0.0001 nM to about 5 nM, and/or the concentrationof the first and second common primers is from about 200 nM to about5000 nM, and/or the concentration of the first and second specificprimers is less than 1 nM, and/or the concentration of the first andsecond common primers is more than 200 nM, and/or the first specificprimer is an omega primer and wherein the second specific primer is anomega primer, and/or the annealing time for the first thermocyclingroutine is from about 30 minute to about 4 hours, and/or the annealingtemperature for the first thermocycling routine is from about 60° C. toabout 75° C., and/or the annealing temperature for the firstthermocycling routine is from about 60° C. to about 72° C., and/or theannealing temperature for the first thermocycling routine is from about65° C. to about 72° C., and/or the second thermocycling routine has from10-50 cycles such as 20-40 cycles, and/or the annealing temperature iswithin 10° C. of the peak polymerase activity of the polymerase, and/orthe polymerase is a polymerase without strand-displacement activity and5′ to 3′ nuclease activity, and/or the polymerase is selected from thegroup consisting of Phusion Hot Start Flex DNA polymerase and Q5® HotStart High-Fidelity DNA Polymerase.

In some embodiments, methods for amplifying two or more different targetnucleic acids are provided. In some embodiments, such methods compriseproviding a set of specific primer pairs wherein each pair comprises afirst specific primer and a second specific primer and is designed for aspecific target nucleic acid, a first common primer, a second commonprimer and a set of target nucleic acids, a polymerase and nucleotides;performing two cycles of a first thermocycling routine comprising andenaturation step, annealing step and an extension step; and performingtwo or more cycles of a second thermocycling routine comprising andenaturation step, annealing step and an extension step therebyamplifying the target nucleic acid. In some such embodiments, theconcentration of the first and second specific primers is 500 fold lessthan that of the first and second common primer, and/or theconcentration of the first and second specific primers is from about0.0001 nM to about 5 nM, and/or the concentration of the first andsecond common primers is from about 200 nM to about 5000 nM, and/or theconcentration of the first and second specific primers is less than 1nM, and/or the concentration of the first and second common primers ismore than 500 nM, and/or the annealing time for the first thermocyclingroutine is from about 30 minute to about 4 hours, and/or the first andsecond specific primers are an omega primers or the first and secondspecific primers are regular specific primers.

In some embodiments, methods for purifying PCR products are provided. Insome embodiments, the methods comprise adding, to a mixture of PCRreaction components comprising target sequences, a first common primerand a second common primer, DNA fragments, polymerase, PCR buffersolution wherein the target sequences are flanked with priming segmentsthat are either identical or complementary to the first common primerand the second common primer, the fragments do not contain primingsegments, and the second common primer comprises a priming segment, amodifier segment and a tag segment; probe grafted beads wherein theprobe has a sequence that is substantially complementary to that of tagsegment and facilitates the capture of the PCR product by the beadsthrough hybridization. In some embodiments of such methods, the modifiersegment is selected from the group consisting of one or more C3 alkylspacers, one or more ethylene glycol spacers, one or morephoto-cleavable spacers, one or more 1′,2′-dideoxyribose, one or moredeoxyuridines or combinations thereof. In some embodiments, the tagsegment comprises at least one binding moiety. In some embodiments, thebinding moiety may be biotin. In some embodiments, the tag segment maycomprise an oligonucleotide and a binding moiety. In some embodiments,the binding moiety may be attached to the 5′ end of the tag segmentoligonucleotide.

In some embodiments, methods for generating surface clusters areprovided. In some embodiments, such methods may comprise amplifying atarget sequence with a first common primer and a second common primerwherein the first common primer comprising a priming segment and thesecond common primer comprising a priming segment, modifier segment andtag segment to produce a PCR product containing a single-stranded tagwherein the PCR product comprises the first strand and the second strandwherein the second strand is connected to the single-stranded tag;providing a substrate wherein the substrate comprises a probe, a firstsurface primer and a second surface primer; applying the PCR product anda guide to the substrate thereby hybridizing the PCR product, the guideand the probe to produce a PCR product/guide/probe complex on thesubstrate surface; ligating the PCR product and the probe therebylinking the PCR product; washing the substrate thereby removing thefirst strand of the PCR product; and, extending the first surface primerand the second surface primer thereby forming surface clusters. In somesuch embodiments, a probe, a first surface primer, and a second surfaceprimer are attached to the substrate. In some embodiments, the probe, afirst surface primer, and a second surface prime further comprise aspacer through which the probe, the first surface primer, and the secondsurface prime may be connected to the substrate surface.

In some embodiments of the methods described herein, the common primermay have a tail segment and a common segment; and/or the specific primerhas a specific segment and a common segment. In some embodiments, theprimer described herein may have a common segment and a specificsegment, wherein the specific segment is comprised of the 3p arm and 5parm and wherein the loop is comprised of the common segment.

In some embodiments, methods for designing a PCR primer are provided. Insome embodiments, such methods may comprise determining a primer lengthto produce a sufficient template association coefficient; determiningprimer 3′ end binding coefficient; determining template associationcoefficient in the presence of folding effect; and, determining primingefficiency by combining association coefficient of variant alleles.

A better understanding of the present invention and of its manyadvantages will be had from the following examples, given by way ofillustration.

EXAMPLES Example I Comparison of Regular and Relay PCR

Lambda DNA (from NEB, Ipswich, Mass.) was used as template, two regularspecific primers and two common primers were used. Phusion Hot StartFlex polymerase Master Mix (from NEB, Ipswich, Mass.) was used. Thecompositions of individual reaction mixtures are listed below. Thereaction in tube 1 is a regular PCR. The reactions in tube 2 and tube 3are relay PCR of utilizing different specific primer concentrations.

TABLE 1 Reaction mixture compositions Units Tube 1 Tube 2 Tube 3Specific Primer nM (per 500 5 0.5 (lambdaPrm1, lambdaPrm2) primer)Common primer nM (per 500 500 (comPrm1, comPrm2) primer)Template-LambdaDNA fM 10 10 10 Phusion Hot Start Flex 2X Master X 1 1 1Mix Total volume μL 25 25 25

Thermo cycling reactions were performed on Thermal Cycler DNA EngineTetrad (from Bio-Rad, Hercules, Calif.). Regular PCR for tube 1 wasconducted using temperature program is shown below.

TABLE 2 PCR temperature program Step Temp (° C.) Time Activation 1 98 30sec Denaturatoin 2 98 15 sec Annealing 3 60 30 sec Extension 4 72 30 secGOTO 2 for 1 time 5 Denaturatoin 6 98 15 sec Extension 7 72 30 sec GOTO6 for 25 times 8 Extension 9 72 10 min Hold 10 4 Forever

Relay PCRs for tubes 2 and 3 were conducted using the temperatureprogram shown below.

TABLE 3 Relay PCR temperature program Step Temp (° C.) Time Activation 198 30 sec Denaturatoin 2 98 15 sec Annealing specific primers 3 60  1 hrExtension 4 72 30 sec GOTO 2 for 1 time 5 Denaturatoin 6 98 15 secInitial common primer anealing 7 60 30 sec Extension 8 72 30 sec GOTO 6for 1 time 9 Denaturatoin 10 98 15 sec Extension 11 72 30 sec GOTO 10for 25 times 12 Extension 13 72 10 min Hold 14 4 Forever

Specific and common primer sequences are list below. All oligonucleotidesequences of this and all following experiments were provided by LCSciences, Houston, Tex. Unless explicitly described, all oligonucleotidesequences are synthesized using conventional synthesis method on CPG(controlled pore glass) substrates (L. J. McBride et al. (1983) “Aninvestigation of several deoxynucleoside phosphoramidites useful forsynthesizing deoxyoligonucleotides” Tetrahedron Letters, 24:245 248).

TABLE 4 Primer sequence list Primer Name Primer Sequence 5′ to 3′lambdaPrm1 GTTCAGAGTTCTACAGTCCGACGATCATACTCCCGA CAATCCCCAC SEQ ID 1lambdaPrm2 CCTTGGCACCCGAGAATTCCAGTATGTCGCAGGTAA AAAGTGC SEQ ID 2 comPrm1AATGATACGGCGACCACCGAGATCTACACGTTCAGA GTTCTACAGTCCGA SEQ ID 3 comPrm2CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA SEQ ID 4

PCR products were analyzed using 3% agarose gel electrophoresis. Theagarose gel was prepared by dissolving 1.2 g of agarose in 40 ml 1×TAEand casted into a gel slab according to the instruction of agarosemanufacture (Grand Island, N.Y.). A 12×7-mm comb was used to createsample loading wells. For gel loading, 1 μL PCR product solution fromeach PCR product tube (of 25 μL) was mixed with 1 μL 6× Blue Gel Loadingbuffer and 4 μL TAE in a PCR tube. The mixtures were thoroughly mixed,spun down, and loaded into the gel loading wells. Electrophoresis wasconducted at 70V for 1 hr 20 min. The gel slab was stained by using SYBRGold by following manufacture instruction (Grand Island, N.Y.). FIG. 16shows an agarose gel electrophoresis image of the experiment. Lanes 1through 3 are the products in tube 1 through 3. Lane L is 50 bp ladderrun. In all three tubes, PCR products of expected sizes were obtained.

The regular PCR in tube 1 included two specific primers and the expectedproduct size is 219 bp. In this reaction, a regular concentration of 500nM was used for both primers. An annealing time of 30 sec was sufficientto have produced the expected product and at expected amount as shown inlane 1 of FIG. 16. The annealing temperature of 60° C. in the first twocycles was determined by the Tms of target specific sections of thespecific primers. In the remaining cycles, combined annealing-extensionsteps at 72° C. were used. The temperature was determined by the Tms ofthe whole specific primers.

The relay PCRs in tubes 2 and 3 involved two specific primers and twocommon primers. The expected product size is 290 bp. Low concentrationsof 5 nM and 0.5 nM were used for specific primers in tube 2 and tube 3,respectively. A long extended annealing time of 1 hr was used in thefirst two cycles to allow hybridizations between the low concentrationspecific primers and corresponding templates. The annealing temperatureof 60° C. in the first two cycles was determined by the Tms of targetspecific sections of the specific primers. Cycles 3 and 4 are designedto add common primer flanks to the cycle 1 and cycle 2 produced targetsequences. The annealing temperature of 60° C. in cycles 3 and 4 wasdetermined by the Tms of common segments of the common primers. In theremaining cycles, combined annealing-extension steps at 72° C. wereused. The temperature was determined by the Tms of the whole commonprimers. In this reaction, a regular concentration of 500 nM was usedfor the common primers. Short annealing time of 30 sec was used for theamplification cycles from cycle 3 till the last cycle. Expected productsat expected amount were observed in the gel image as shown in lanes 2and 3 of FIG. 14.

Control experiments were performed. The first control experimentinvolves the two common primers (comPrm1 and comPrm2) at 500 nM each,lambda DNA at 10 fM, and Phusion Hot Start Flex polymerase Master Mix(from NEB, Ipswich, Mass.). The second control experiment involves thetwo specific primers (lambdaPrm1 and lambdaPrm2) at 50 nM each, the twocommon primers (comPrm1 and comPrm2) at 500 nM each, without lambda DNA,and Phusion Hot Start Flex polymerase Master Mix (from NEB, Ipswich,Mass.). Both control experiments were carried out using the relay PCRtemperature program shown above. No product was observed in eitherreaction.

Example II Relay PCR Using Omega Primer for Amplification of HumanGenomic DNA

Human DNA was used as template, two regular specific primers and twocommon primers were used. Phusion Hot Start Flex polymerase Master Mix(from NEB, Ipswich, Mass.) was used. Six pairs of specific primers wereused to individually amplify six target sequences. A pair of commonprimers was used in combination with each pair of the specific primers.Total six amplification reactions plus one no-specific primer controlwere conducted in six tubes. The compositions of the reaction mixturesare listed below.

TABLE 5 Reaction mixture compositions Units Tube 1-6 Tube 7 SpecificPrimer (specPrm1, nM (per primer) 1 specPrm2) Common primer (comPrm1, nM(per primer) 500 500 comPrm2) Template - human gDNA fM 2 2 Phusion HotStart Flex 2X X 1 1 Master Mix Total volume μL 25 25

Thermo cycling reactions were performed on Thermal Cycler DNA EngineTetrad (from Bio-Rad, Hercules, Calif.) using the temperature programshown below.

TABLE 6 Relay PCR temperature program Step Temp (° C.) Time Activation 198  5 min Denature 2 98  15 sec Specific primer annealing 3 65 120 minExtension 1 4 68 120 sec Extension 2 5 72 120 sec GOTO 2 for 1 time 6Denature 7 98  15 sec Initiatial common primer anealing 8 60  30 secExtension 1 9 68 120 sec Extension 2 10 72 120 sec GOTO 7 for 1 time 11Denature 12 98  15 sec Extension 1 13 68 120 sec Extension 2 14 72 120sec GOTO 12 for 25 times 15 Extension 16 72  10 min Hold 17 4 Forever

Specific primer sequence information is listed below.

TABLE 7 Specific primer sequence information index prmName rxtTubetgtChr tgtStrand prmStrand tgtStart/End tgtLength ampliconLength 1TP53_31_59_tile03_O1 1 chr17 + − 7578615 2 TP53_31_59_tile03_O2 1chr17 + + 7578439 176 293 3 TP53_31_59_tile01_O1 2 chr17 + − 7578353 4TP53_31_59_tile01_O2 2 chr17 + + 7578162 191 308 5 PIK3CA12_tile01_O1 3chr3 + − 178938982 6 PIK3CA12_tile01_O2 3 chr3 + + 178938809 173 290 7KRAS1_tile01_O1 4 chr12 − + 25378405 8 KRAS1_tile01_O2 4 chr12 − −25378595 190 307 9 APC1_tile01_O1 5 chr5 − + 112173871 10 APC1_tile01_O25 chr5 − − 112174042 171 288 11 APC2_tile01_O1 6 chr5 − + 112174557 12APC2_tile01_O2 6 chr5 − − 112174730 173 290

TABLE 8 Primer sequence list index prmSeq  1CGCATGTTTGTTTCTTTGCTGCCGTCTTCCAGGTTCAGAGTTCTACAGTCCGACGATCTTGCTTTATCTGTTCACTTGTG  SEQ ID 5  2ACAACCTCCGTCATGTGCTGTGACTGCTCCTTGGCACCCGAGAATTCCATGTAGATGGCCATGGC SEQ ID 6  3GCGATGGTGAGCAGCTGGGGCTGGGTTCAGAGTTCTACAGTCCGACGATCAGAGACGACAGGGC SEQ ID 7  4CCCTTAACCCCTCCTCCCAGAGACCCCACCTTGGCACCCGAGAATTCCAGTTGCAAACCAGACCT SEQ ID 8  5GGGCTTCTAAACAACTCTGCCCCACTGCAGGTTCAGAGTTCTACAGTCCGACGATCTGAAAAGAGTCTCAAACACAAAC  SEQ ID 9  6CTTTTAGATCTGAGATGCACAATAAAACAGTTAGCCAGAGGTTCCTTGGCACCCGAGAATTCCATGGCCTGCTTTTGG  SEQ ID 10  7CCAAAAGCAGTACCATGGACACTGGATTAAGAAGCAATGGTTCAGAGTTCTACAGTCCGACGATCCCCTCTCAAGAGACAAAAA CA SEQ ID 11  8AACAGTAGACACAAAACAGGCTCAGGACTTAGCAACCTTGGCACCCGAGAATTCCAGAAGTTATGGAATTCCTTTTATTGAAACA  SEQ ID 12  9AGATAGAAGTTTGGAGAGAGAACGCGGAATTGGTCTAGTTCAGAGTTCTACAGTCCGACGATCGGCAACTACCATCCAGC  SEQ ID 13 10GGGCAGCAGAGCTTCTTCTAAGTGCATTTCTCTCACCTTGGCACCCGAGAATTCCATCTGTCACACAATGTAATTCAGT  SEQ ID 14 11CCTGTTTATACTGAGAGCACTGATGATAAACACCTCAAGTTGTTCAGAGTTCTACAGTCCGACGATCCCAACCACATTTTGGAC AG SEQ ID 15 12GTTGGTCTCTCTTCTTCTTCATGCTGTTCTTCTTCAGAGTACCTTGGCACCCGAGAATTCCAACGTTCACTATAATTGGTAGGC SEQ ID 16

The common primers that were used were the same as that of Experiment I.PCR products were analyzed using the same agarose gel electrophoresismethod as that of Experiment I. FIG. 16 shows the gel image. Lane 1through lane 6 shows the products from the six individual PCR reactions.Lane 7 shows the result of no-specific primer control run. Lane L is aDNA ladder showing the sizes (in base pair or bp) of correspondingmarkers. In all six tubes, PCR products of expected sizes were obtained.The following table shows the relative signals of product bands in thegel image. The standard deviation of the relative signals is 0.191. Thegel signal values were extracted from the gel image using Array-Pro®analyzer software (from MediaCybernetics, Rockville, Md.). The relativesignals are derived by dividing signals by signal median. No detectablePCR product is observed in the no-specific primer experiment of lane 7.

TABLE 9 Relative signals of product bands Signal Relative rxtTubeTgtName (mean) Signal 1 TP53-T3 9,049 0.68 2 TP53-T1 13,948 1.04 3PIK3CA12-T1 16,866 1.26 4 KRAS1-T1 11,912 0.89 5 APC1-T1 13,707 1.02 6APC2-T1 13,094 0.98

Example III Multiplex PCR to Amplify all Six Targets of Experiment II ina Single Tube

The same six pairs of omega primers and one pair of common primers asthat of Experiment II were used. The composition of the reactionmixtures is shown below.

TABLE 10 Reaction mixture compositions Units Tube 1 Specific Primer (12primers) nM (per primer) 1 Common primer (comPrm1, comPrm2) nM (perprimer) 500 Template - human gDNA fM 2 Phusion Hot Start Flex 2X MasterMix X 1 Total volume μL 25

Thermo cycling reactions were performed on Thermal Cycler DNA EngineTetrad (from Bio-Rad, Hercules, Calif.) using the temperature programshown below.

TABLE 11 Relay PCR temperature program Step Temp (° C.) Time Activation1 98 5 min Denature 2 98 15 sec Specific primer annealing 3 65 120 minExtension 1 4 68 60 sec Extension 2 5 72 60 sec GOTO 2 for 1 time 6Denature 7 98 15 sec Initiatial common primer anealing 8 60 30 secExtension 1 9 68 60 sec Extension 2 10 72 60 sec GOTO 7 for 1 time 11Denature 12 98 15 sec Extension 1 13 68 60 sec Extension 2 14 72 60 secGOTO 12 for 25 times 15 Extension 16 72 10 min Hold 17 4 Forever

PCR products were analyzed using the same agarose gel electrophoresismethod as that of Experiment I. FIG. 18A shows the gel image. Lane 1shows the product of the multiplex PCR. Lane L is a DNA ladder showingthe sizes (in base pair or bp) of corresponding markers. The sizedistribution of the PCR product is in the expected size range of 288 to308 bp. The multiplex PCR product was also analyzed by parallelsequencing using HiSeq 2000 (from Illumina, San Diego, Calif.). All sixexpected amplicons produced sequencing reads. The following table showsthe read number distribution of individual amplicons of the targetsequences. In the table, read fraction is calculated by dividing theread number of each target sequence by the total number of reads.Relative read fraction is calculated by dividing the read fraction bymedian value of the read fraction. All 6 amplicons had read number above20% of median read numbers. The standard deviation of the relative readfraction is 0.512. FIG. 18B shows a scatter plot of the sequencing readnumber distribution of the 6 expected amplicons.

TABLE 12 Sequencing derived read number distribution of individualamplicons Relative Read Read Read rxtTube TgtName Number FractionFraction 1 TP53-T3 152,832 0.12 0.86 2 TP53-T1 211,454 0.16 1.14 3PIK3CA12-T1 354,681 0.28 2.00 4 KRAS1-T1 135,307 0.10 0.71 5 APC1-T1271,654 0.21 1.50 6 APC2-T1 134,248 0.10 0.71

Example IV Multiplex PCR Using Omega Primers to Amplify 44 Targets in aSingle Tube

A multiplex relay PCR using omega primer in a single tube was performedto capture and amplify 44 targets in human genomic DNA. The amplicondistribution in the PCR product was obtained by sequencing using HiSeq2000 (from Illumina, San Diego, Calif.) sequencer. 88 omega primers weredesigned to capture 44 specific target regions of human genome accordingto the disclosed primer design and computation methods. Genome assemblyversion GRCh37/hg19 was used in the used in the target/primer design.The information of the captured regions is listed in the followingtable. In the table, we call the capture region including 3p armsegments of omega primers as “probe” (prb). The last two columns of thetable list the indexes of paired primers. The omega specific primersequences were designed according to the design methods of thisdisclosure and the common primers were the same as those of ExperimentI.

TABLE 13 List of the captured regions index prbIndex ampliconID geneNamechr prbStart prbEnd prm1Idx prm2Idx 1 prb_2ERBB4_3_4_chr2_212530002_175_140509-1_t2 ERBB4_3_4 chr2 212530002212530176 prm_3 prm_4 2 prb_6 ERBB4_9_chr2_212589687_190_140509-1_t2ERBB4_9 chr2 212589687 212589876 prm_11 prm_12 3 prb_8ERBB4_11_chr2_212812087_198_140509-1_t2 ERBB4_11 chr2 212812087212812284 prm_15 prm_16 4 prb_10 VHL3_5_chr3_10188197_183_140509-1_t2VHL3_5 chr3 10188197 10188379 prm_19 prm_20 5 prb_11VHL6_8_chr3_10191403_171_140509-1_t2 VHL6_8 chr3 10191403 10191573prm_21 prm_22 6 prb_15 PIK3CA4_11_chr3_178936054_189_140503-1_t2PIK3CA4_11 chr3 178936054 178936242 prm_29 prm_30 7 prb_16PIK3CA12_chr3_178938706 188_140503-1_t2 PIK3CA12 chr3 178938706178938893 prm_31 prm_32 8 prb_18PIK3CA13_20_chr3_178952024_175_140503-1_t2 PIK3CA13_20 chr3 178952024178952198 prm_35 prm_36 9 prb_20 APC2_chr5_112174500_190_140503-1_t2APC2 chr5 112174500 112174689 prm_39 prm_40 10 prb_22APC3_42_chr5_112175257_185_140503-1_t2 APC3_42 chr5 112175257 112175441prm_43 prm_44 11 prb_24 APC3_42_chr5_112175519_186_140503-1_t2 APC3_42chr5 112175519 112175704 prm_47 prm_48 12 prb_26APC3_42_chr5_112175778_187_140503-1_t2 APC3_42 chr5 112175778 112175964prm_51 prm_52 13 prb_28 EGFR1_chr7_55211035_171_140503-1_t2 EGFR1 chr755211035 55211205 prm_55 prm_56 14 prb_29EGFR2_chr7_55221780_171_140503-1_t2 EGFR2 chr7 55221780 55221950 prm_57prm_58 15 prb_30 EGFR3_chr7_55232965_170_140503-1_t2 EGFR3 chr7 5523296555233134 prm_59 prm_60 16 prb_31 EGFR4_chr7_55241618_180_140503-1_t2EGFR4 chr7 55241618 55241797 prm_61 prm_62 17 prb_32EGFR9_chr7_55242352_190_140503-1_t2 EGFR9 chr7 55242352 55242541 prm_63prm_64 18 prb_33 EGFR44_chr7_55248978_177_140503-1_t2 EGFR44 chr755248978 55249154 prm_65 prm_66 19 prb_34EGFR54_chr7_55259367_173_140503-1_t2 EGFR54 chr7 55259367 55259539prm_67 prm_68 20 prb_37 PTEN3_chr10_89685268_190_140503-1_t2 PTEN3 chr1089685268 89685457 prm_73 prm_74 21 prb_38PTEN4_chr10_89711829_184_140503-1_t2 PTEN4 chr10 89711829 89712012prm_75 prm_76 22 prb_40 PTEN7_chr10_89717703_187_140503-1_t2 PTEN7 chr1089717703 89717889 prm_79 prm_80 23 prb_43ATM2_chr11_108119751_197_140509-1_t2 ATM2 chr11 108119751 1081199475prm_85 prm_86 24 prb_44 ATM3_chr11_108123509_218_140509-1_t2 ATM3 chr11108123509 108123726 prm_87 prm_88 25 prb_47ATM7_chr11_108170341_193_140509-1_t2 ATM7 chr11 108170341 108170533prm_93 prm_94 26 prb_49 ATM10_chr11_108173612_206_140509-1_t2 ATM10chr11 108173612 108173817 prm_97 prm_98 27 prb_53ATM15_chr11_108205687_168_140509-1_t2 ATM15 chr11 108205687 108205854prm_105 prm_106 28 prb_54 ATM16_chr11_108206455_168_140509-1_t2 ATM16chr11 108206455 108206622 prm_107 prm_108 29 prb_56ATM18_chr11_108225561_176_140509-1_t2 ATM18 chr11 108225561 108225736prm_111 prm_112 30 prb_57 ATM19_chr11_108236033_185_140509-1_t2 ATM19chr11 108236033 108236217 prm_113 prm_114 31 prb_61FLT3_1_chr13_28592542_171_140509-1_t2 FLT3_1 chr13 28592542 28592712prm_121 prm_122 32 prb_62 FLT3_13_chr13_28602179_187_140509-1_t2 FLT3_13chr13 28602179 28602365 prm_123 prm_124 33 prb_64FLT3_22_chr13_28610028_170_140509-1_t2 FLT3_22 chr13 28610028 28610197prm_127 prm_128 34 prb_66 BRCA2_chr13_32907302_179_140509-1_t2 BRCA2chr13 32907302 32907480 prm_131 prm_132 35 prb_69BRCA2_chr13_32912508_181_140509-1_t2 BRCA2 chr13 32912508 32912688prm_137 prm_138 36 prb_70 BRCA2_chr13_32920892_196_140509-1_t2 BRCA2chr13 32920892 32921087 prm_139 prm_140 37 prb_74BRCA2_chr13_32954042_188_140509-1_t2 BRCA2 chr13 32954042 32954229prm_147 prm_148 38 prb_75 BRCA2_chr13_32970885_203_140509-1_t2 BRCA2chr13 32970885 32971087 prm_149 prm_150 39 prb_76BRCA2_chr13_32972487_199_140509-1_t2 BRCA2 chr13 32972487 32972685prm_151 prm_152 40 prb_82 TP53_60_chr17_7579298_187_140503-1_t2 TP53_60chr17 7579298 7579484 prm_163 prm_164 41 prb_85ERBB2_4_chr17_37880908_176_140509-1_t2 ERBB2_4 chr17 37880908 37881083prm_169 prm_170 42 prb_86 ERBB2_13_chr17_37881235_220_140509-1_t2ERBB2_13 chr17 37881235 37881454 prm_171 prm_172 43 prb_87BRCA1_chr17_41243526_181_140509-1_t2 BRCA1 chr17 41243526 41243706prm_173 prm_174 44 prb_92 BRCA1_chr17_41267714 178_140509-1_t2 BRCA1chr17 41267714 41267891 prm_183 prm_184

The composition of the reaction mixtures is shown below.

TABLE 14 Reaction mixture compositions Units Tube A Tube B Tube CSpecific Primer (88 primers) nM (per 1 0.2 0.04 primer) Common primer(comPrm1, nM (per 500 500 500 comPrm2) primer) Template - human gDNA fM2 2 2 Phusion Hot Start Flex 2X X 1 1 1 Master Mix Total volume μL 25 2525

Thermo cycling reactions were performed on Thermal Cycler DNA EngineTetrad (from Bio-Rad, Hercules, Calif.) using the temperature programshown below.

TABLE 15 Relay PCR temperature program Step Temp (° C.) Time Activation1 98 5 min Denature 2 98 15 sec Specific primer annealing 3 65 120 minExtension 1 4 68 60 sec Extension 2 5 72 60 sec GOTO 2 for 1 time 6Denature 7 98 15 sec Initiatial common primer anealing 8 60 30 secExtension 1 9 68 60 sec Extension 2 10 72 60 sec GOTO 7 for 1 time 11Denature 12 98 15 sec Extension 1 13 68 60 sec Extension 2 14 72 60 secGOTO 12 for 25 times 15 Extension 16 72 10 min Hold 17 4 Forever

The multiplex PCR products were analyzed by parallel sequencing usingHiSeq 2000 (from Illumina, San Diego, Calif.). FIG. 19 shows thesequencing measurement results of amplicon read number distributions.Figures A, B, and C plot the results obtained by using specific omegaprimer concentrations of 1 nM, 0.2 nM, and 0.04 nM per primer,respectively. All 44 designed target regions produced amplicons asobserved in the sequencing result. More than 95% the amplicons had readnumber above 20% of median read numbers in PCR products from all threespecific omega primer concentrations.

Example V Relay PCR and Annealing Time

An experiment was conducted to reveal the relay PCR yield dependence onphase I specific primer annealing time at specific primer concentrationsof 5 nM and 1 nM, respectively. The composition of the reaction mixturesis shown below.

TABLE 16 Reaction mixture compositions Units Tubes 1-5 Tubes 6-10Specific Primer nM (per primer) 5 1 (APC1_R1, APC1_R2) Common primer nM(per primer) 500 500 (comPrm1, comPrm2) Template—human gDNA fM 2 2Phusion Hot Start X 1 1 Flex 2X Master Mix Total volume μL 25 25Specific primer min 10-240 10-240 annealing time

Specific primer sequences are list below. Common primers were the sameas those of Example I.

TABLE 17 List of primer sequences Primer Name Primer Sequence 5′ to 3′APC1_R1 GTTCAGAGTTCTACAGTCCGACGATCGAGAG AACGCGGAATTGGTCTAGGCA SEQ ID 17APC1_R2 CCTTGGCACCCGAGAATTCCAAGTGGTAGAC CCAGAACTTCTGTCTTCCT SEQ ID 18

Thermo cycling reactions were performed on Thermal Cycler DNA EngineTetrad (from Bio-Rad, Hercules, Calif.) using the temperature programshown below.

TABLE 18 Relay PCR temperature program Temp Step (° C.) Time Activation1 98 5 min Denature 2 98 15 sec Specific primer annealing 3 65 10, 30,60, 120, 240 min Extension 4 72 30 sec GOTO 2 for 1 time 5 Denature 6 9815 sec Initiatial common 7 60 30 sec primer anealing Extension 8 72 30sec GOTO 6 for 1 time 9 Denature 10 98 15 sec Extension 11 72 30 secGOTO 10 for 25 times 12 Extension 13 72 10 min Hold 14 4 Forever

PCR products were analyzed using the same agarose gel electrophoresismethod as that of Experiment I. In all 10 tubes, PCR products ofexpected size (288 bp) were obtained. The following table shows thesignals and relative signals of product bands extracted from the gelimage. The gel signal values were extracted by using Array-Pro® analyzersoftware (from MediaCybernetics, Rockville, Md.). The relative signalsare derived by dividing corresponding signals by the maximum signalwithin the same specific primer concentration. From the data, it isnoted that at high specific primer concentration of 5 nM, relativesignal rapidly reaches a high relative signal of 0.7 within a shortspecific primer annealing time of 10 min. However, at a low specificconcentration of 1 nM, a significantly extended specific primerannealing time of 60 min or more is required to obtain a relative signalapproaching 1.

TABLE 19 Product band signals Specific primer Specific annealing timeSignal Relative primer (min) (mean) Signal conc (nM) 10 9,488 0.70 5 3013,607 1.00 5 60 8,175 0.60 5 120 9,050 0.67 5 240 5,795 0.43 5 10 20.00 1 30 2,211 0.37 1 60 4,973 0.84 1 120 5,887 1.00 1 240 5,909 1.00 1

Example VI Multiplex Relay PCR Using Microarray Synthesized SpecificPrimer Precursors

An experiment was conducted to practice multiplex relay PCR usingmicroarray synthesized specific primer precursors. The method of thisexperiment has been described in the exemplary embodiment relating toFIG. 9 of this disclosure.

A group of 204 omega primer precursor sequences for capturing 102specific target regions in human genome were designed according to thedesign methods of this disclosure. All omega primer precursor sequenceshave the same 5′ and 3′ flank segments for PCR amplification use.Following table lists two exemplary omega primer precursor sequences (2out of 204) and two preparation primer sequences. As describe earlier inthis disclosure, preparation primer prepPrm2 has a dU at its 3′terminal. The 204 omega primer precursor sequences were synthesizedusing microarray synthesis method and the two preparation primers weresynthesized using conventional oligonucleotide synthesis method. All 204omega primer precursor sequences were synthesized in parallel andprovided by the manufacture LC Sciences (Houston, Tex.) in a mixtureform and in a single tube.

TABLE 20 List of primer sequences Sequence Name Sequence 5′ to 3′MPL1_2_tile01_ GAGCTTCGGTTCACGCAATGCCGAAGTCTGACC prm1_tube1CTTTTTGTCTCCTAGCCGTTCAGAGTTCTACAG TCCGACGATCTGGATCTCCTTGGTAGTTGATCCGGTCCTAGGCA SEQ ID 19 MPL1_2_tile01_ GAGCTTCGGTTCACGCAATGACGGAGATCTGGGprm2_tube1 GTCACAGAGCGACCTTGGCACCCGAGAATTCCAACCAAGAATGCCTAGTTGATCCGGTCCTAGGCA SEQ ID 20 prepPrm1GAGCTTCGGTTCACGCAATG SEQ ID 21 prepPrm2TGCCTAGGACCGGATCAAC/dU/ SEQ ID 22

The omega primer precursor sequence mixture was first amplified by PCRusing Taq hot start 2X master mix (from NEB, Ipswich, Mass.). The PCRreaction mixture composition is shown below.

TABLE 21 Reaction mixture compositions Units Tube 1 Common primer(prepPrm1, prepPrm2) nM (per primer) 500 Template-omega primer precursormix pM 1 Taq Hot Start 2X Master Mix X 1 Total volume μL 25

Thermo cycling reactions were performed on Thermal Cycler DNA EngineTetrad (from Bio-Rad, Hercules, Calif.). PCR temperature program isshown below.

TABLE 22 PCR temperature program Step Temp (° C.) Time Activation 1 95 5min Denaturatoin 2 95 30 sec Annealing 3 60 60 sec Extension 4 68 60 secGOTO 2 for 10 time 5 Extension 6 72 10 min Hold 7 4 Forever

PCR products were analyzed using the same agarose gel electrophoresismethod as that of Experiment I. FIG. 20A lane 1 shows the agarose gelelectrophoresis image of the PCR product. PCR product of the expectedsize distribution of 95-130 bp with a median size of 110 bp wasobserved. The PCR product of the omega primer precursors was purifiedusing PCR purification beats (Agencourt AMPure XP system from BeckmanCoulter, Brea, Calif.) by following manufacture instruction.Concentration of the purified PCR product was measured by Bioanalyzerfrom Agilent (Santa Clara, Calif.).

Then, dU in the PCR product was removed using UDG/EDA process. A UDGdigestion solution is prepared according to the following table. UDG andUDG buffer were purchased from NEB (Ipswich, Mass.). The solution wasincubated at 37° C. for 60 minutes. Then, 2 μL 200 mM EDA (fromSigma-Aldrich, St. Louis, Mo.) was added into the solution and incubatedat 37° C. for another 60 minutes.

TABLE 23 Reaction mixture composition Units Tube 1 Total volume μL 10PCR product μL 8 UDG (5U/ul) μL 1 UDG buffer, 10× μL 1

Relay PCR using the amplified and dU removed specific primer precursorswas carried out using human genomic DNA as template and Phusion HotStart Flex polymerase Master Mix (from NEB, Ipswich, Mass.) aspolymerase. The compositions of individual reaction components arelisted below. Reaction in tube 1 is a negative control without addingthe specific primer precursor. Reaction in tube 2 is the test for therelay PCR.

TABLE 24 Reaction mixture composition Units Tube 1 Tube 2 Specificprimer precursors nM (total) 1.4 prepPrm1 nM 200 200 Common primers(comPrm1, nM (per primer) 500 500 comPrm2) Template-Human gDNA fM 2 2Phusion Hot Start Flex 2X X 1 1 Master Mix Total volume μL 25 25

Thermo cycling reactions were performed on Thermal Cycler DNA EngineTetrad (from Bio-Rad, Hercules, Calif.). PCR temperature program isshown below.

TABLE 25 Relay PCR temperature program Step Temp (° C.) Time Activation1 98 5 min Annealing 2 68 1 min Extension 3 72 1 min Denature 4 98 15sec Annealing 5 65 120 min Extension 6 72 30 sec GOTO 4 for 1 time 7Denature 8 98 15 sec Annealing 9 60 30 sec Extension 10 72 30 sec GOTO 8for 1 time 11 Denature 12 98 15 sec Extension 13 72 30 sec GOTO 12 for25 times 14 Extension 15 72 10 min Hold 16 4 Forever

Products of the relay PCR were analyzed using the same agarose gelelectrophoresis method as that of Experiment I. FIG. 20B shows theimages of the PCR products. Lane L is a DNA ladder showing the sizes (inbase pair or bp) of corresponding markers. Lane 1 shows the result ofthe negative control of tube 1. No PCR product is observed in thenegative control (lower bands are due to unused primers). Lane 2 isloaded with the PCR product from tube 2. An expected PCR product sizedistribution of 295-399 bp with a median of 317 bp was observed.

Example VII Multiplex Relay PCR Using Specific Primers Prepared by PCRAmplification

An experiment was conducted to practice multiplex relay PCR usingspecific primers prepared by PCR amplification. The method of thisexperiment has been described in the exemplary embodiment relating toFIG. 11 of this disclosure.

Omega primers for capturing a target region in human genome of genomeassembly version GRCh37/hg19 were designed according to the designmethods of this disclosure. The captured target region belongs to anexon region of APC1 gene in chromosome 5 and with starting position of112,173,776 and ending position of 112,173,955. The following tablelists the oligonucleotide sequences used in this experiment. Preparationprimers prepPrm1 and prepPrm2 are designed to PCR amplify specificprimer templates. A restriction recognition site GCTCTTC is embedded inprepPrm2 sequence to facilitate restriction cut of the PCR product byrestriction nuclease BspQI. Specific primer templatesAPC1_chr5_(—)112173790_p1 and APC1_chr5_(—)112173944_p2 containing 5′ aswell as 3′ flanking segments are designed to be PCR amplified byprepPrm1 and prepPrm2. The mid-section of the specific primer templatesare designed as omega primers. Specific primersAPC1_chr5_(—)112173790_p1_no3pFlank andAPC1_chr5_(—)112173944_p2_no3pFlank have the same omega primer designsas that of APC1_chr5_(—)112173790_p1 and APC1_chr5_(—)112173944_p2 butdo not have 3′ flank segments. They are active specific primers and wereused as references to be compared with specific primer template derivedprimers for capturing the target region in relay PCR reactions. PrimerscomPrm1 and comPrm2 were designed as common primers of relay PCR. Theoligonucleotides in the following table were synthesized usingconventional method and were provided by LC Sciences (Houston, Tex.).

TABLE 26 List of primer sequences Sequence Name Sequence 5′ to 3′prepPrm1 TTTTCGCGTTAGTATCCGACCGATCTACGTAGCG SEQ ID 23 prepPrm2TTTTGACCGTACTATCGAACCGTCGTACTAGCTC TTCGCGT SEQ ID 24 APC1_chr5_ATCCGACCGATCTACGTAGCGGGCAACATGACTG 112173790_p1TCCTTTCACCATATTTGAATACTCGTTCAGAGTT CTACAGTCCGACGATCACAGTGTTACCCAGCACGCGAAGAGCTAGTACGACGG SEQ ID APC1_chr5_ ATCCGACCGATCTACGTAGCGGGTATGAATGGCT112173944_p2 GACACTTCTTCCATGACTTTCCTTGGCACCCGAGAATTCCAGGCAATCTGGGCACGCGAAGAGCTAGT ACGACGG SEQ ID 26 APC1_chr5_TCCGACCGATCTACGTAGCGGGCAACATGACTGT 112173790_CCTTTCACCATATTTGAATACTCGTTCAGAGTTC p1_no3pFlankTACAGTCCGACGATCACAGTGTTACCCAGC  SEQ ID 27 APC1_chr5_TCCGACCGATCTACGTAGCGGGTATGAATGGCTG 112173944_ACACTTCTTCCATGACTTTCCTTGGCACCCGAGA p2_no3pFlankATTCCAGGCAATCTGGGC SEQ ID 28 comPrm1 AATGATACGGCGACCACCGAGATCTACACATGATGACACACGTTCAGAGTTCTACAGTCCGA  SEQ ID 29 comPrm2CAAGCAGAAGACGGCATACGAGATGAATGATAGT GACTGGAGTTCCTTGGCACCCGAGAATTCCA SEQ ID 30

To amplify the specific primer templates, the two specific primertemplates were mixed in equal concentration together with PCR componentsas shown in the table below. Hot start Phusion polymerase from (fromNEB, Ipswich, Mass.) was used in this reaction.

TABLE 27 Reaction mixture composition Units Tube 1 Preparation primer(prepPrm1, prepPrm2) nM (per primer) 500 Template(APC1_chr5_112173790_p1) fM 20 Template (APC1_chr5_112173944_p2) fM 20Hot Start Phusion Flex 2X Master Mix X 1 Total volume μL 25

Thermo cycling reactions were performed on Thermal Cycler DNA EngineTetrad (from Bio-Rad, Hercules, Calif.). PCR temperature program isshown below.

TABLE 28 PCR temperature program Step Temp (° C.) Time Activation 1 98 5min Denature 2 98 15 sec Annealing 3 70 30 sec Extension 4 72 60 secGOTO 2 for 1 time 5 Denature 6 98 15 sec Annealing-Extension 7 72 60 secGOTO 6 for 19 time 8 Extension 9 72 10 min Hold 10 4 Forever

The PCR product was analyzed using the same agarose gel electrophoresismethod as that of Experiment I. FIG. 21A lane 1 shows the gel image ofthe PCR products. The predicted sizes of the two PCR products are 153 bpand 141 bp, respectively, and the gel band positions agree with theprediction. The PCR product was purified using PCR purification beats(Agencourt AMPure XP system from Beckman Coulter, Brea, Calif.) byfollowing manufacture instruction. The concentration of the purified PCRproduct was measured by NanoDrop spectrometer (from NanoDrop products,Wilmington, Del.).

Restriction enzyme digestion was then applied to the PCR product usingthe reaction compositions shown in the table below. Restriction enzymeBspQI along with 10× Cutsmart buffer was purchased from NEB (Ipswich,Mass.). The digestion reaction was carried out at the enzyme manufacturesuggested condition of 50° C. for 30 minutes. The digestion product wasthen purified using PCR purification beats (Agencourt AMPure XP systemfrom Beckman Coulter, Brea, Calif.) by following manufactureinstruction. The purified digestion product was analyzed using the sameagarose gel electrophoresis method as that of Experiment I. FIG. 21Blane 2 shows the gel image of the product. The predicted sizes of thetwo digestion products are 132 bp and 120 bp, respectively, and the gelband positions agree with the prediction. FIG. 21B lane 1 shows theoriginal PCR product before restriction enzyme digestion.

TABLE 29 Reaction mixture composition Units Tube 1 Purified PCR product(30 ng/uL) μL 20 10x Cutsmart buffer (NEB) μL 2.5 BspQI (NEB) μL 1 H2OμL 1.5 Total volume μL 25

The restriction enzyme digested product was then subject to Lambdaexonuclease digestion to produce single strand specific primer sequencesusing the reaction compositions shown in the table below. Lambdaexonuclease along with a 10× reaction buffer was purchased from NEB(Ipswich, Mass.). The digestion reaction was carried out at the enzymemanufacture suggested condition of 37° C. for 30 minutes. The digestionproduct was then purified using PCR purification beats (Agencourt AMPureXP system from Beckman Coulter, Brea, Calif.) by following manufactureinstruction. We call the product enzymatically prepared specificprimers.

TABLE 30 Reaction mixture composition Units Tube 1 Restriction digestedproduct (20 ng/uL) μL 8 10X Lambda exonuclease reaction buffer μL 1Lambda exonuclease (1000 U/ml) μL 1 Total volume μL 10

Relay PCR reactions using the enzymatically prepared specific primerswas carried out using human genomic DNA as template and Phusion HotStart Flex polymerase Master Mix (from NEB, Ipswich, Mass.) aspolymerase. The compositions of individual reaction components arelisted in the table below. Reaction in tube 1 is the positive controlusing reference specific primers APC1_chr5_(—)112173790_p1_no3pFlank andAPC1_chr5_(—)112173944_p2_no3pFlank. Reaction in tube 2 is the testusing enzymatically prepared specific primers. Reaction in tube 3 is anegative control without adding genomic DNA.

TABLE 31 Reaction mixture composition Units Tube 1 Tube 2 Tube 3Specific primer nM (per primer) 1 (reference) Specific primer nM (perprimer) 1 1 (enzymatically prepared) Common primers nM (per primer) 500500 500 (comPrm1, comPrm2) Template-Human gDNA fM 1 1 Hot Start PhusionFlex X 1 1 1 2X Master Mix Total volume μL 25 25 25

Thermo cycling reactions were performed on Thermal Cycler DNA EngineTetrad (from Bio-Rad, Hercules, Calif.). PCR temperature program isshown below.

TABLE 32 Relay PCR temperature program Step Temp (° C.) Time Activation1 98 5 min Denature 2 98 15 sec Anneal Omega primers 3 65 120 minExtension 1 4 68 60 sec Extension 2 5 72 60 sec GOTO 2 for 1 time 6Denature 7 98 15 sec Initiate lib primer anealing 8 60 30 sec Extension1 9 68 60 sec Extension 2 10 72 60 sec GOTO 7 for 1 time 11 Denature 1298 15 sec Extension 1 13 68 60 sec Extension 2 14 72 60 sec GOTO 12 for27 times 15 Extension 16 72 10 min Hold 17 4 Forever

Products of the relay PCR were analyzed using the same agarose gelelectrophoresis method as that of Experiment I. FIG. 21C shows theimages of the PCR products. Lane L is a DNA ladder showing the sizes (inbase pair or bp) of corresponding markers. Lane 1 shows the result ofthe positive control of tube 1 using conventionally synthesizedreference specific primers. Lane 2 is the test from tube 2 usingenzymatically prepared specific primers. The same product sizes around300 bp are observed in the positive control of lane 1 and in the test oflane 2. The size is consistent with the predicted size of 312 bp. Lane 3is the result of negative control from tube 3 which shows no productaround 300 bp.

Within this disclosure, any indication that a feature is optional isintended provide adequate support (e.g., under 35 U.S.C. 112 or Art. 83and 84 of EPC) for claims that include closed or exclusive or negativelanguage with reference to the optional feature. Exclusive languagespecifically excludes the particular recited feature from including anyadditional subject matter. For example, if it is indicated that A can bedrug X, such language is intended to provide support for a claim thatexplicitly specifies that A consists of X alone, or that A does notinclude any other drugs besides X. “Negative” language explicitlyexcludes the optional feature itself from the scope of the claims. Forexample, if it is indicated that element A can include X, such languageis intended to provide support for a claim that explicitly specifiesthat A does not include X. Non-limiting examples of exclusive ornegative terms include “only,” “solely,” “consisting of,” “consistingessentially of,” “alone,” “without”, “in the absence of (e.g., otheritems of the same type, structure and/or function)” “excluding,” “notincluding”, “not”, “cannot,” or any combination and/or variation of suchlanguage.

Similarly, referents such as “a,” “an,” “said,” or “the,” are intendedto support both single and/or plural occurrences unless the contextindicates otherwise. For example “a dog” is intended to include supportfor one dog, no more than one dog, at least one dog, a plurality ofdogs, etc. Non-limiting examples of qualifying terms that indicatesingularity include “a single”, “one,” “alone”, “only one,” “not morethan one”, etc. Non-limiting examples of qualifying terms that indicate(potential or actual) plurality include “at least one,” “one or more,”“more than one,” “two or more,” “a multiplicity,” “a plurality,” “anycombination of,” “any permutation of,” “any one or more of,” etc. Claimsor descriptions that include “or” between one or more members of a groupare considered satisfied if one, more than one, or all of the groupmembers are present in, employed in, or otherwise relevant to a givenproduct or process unless indicated to the contrary or otherwise evidentfrom the context.

Where ranges are given herein, the endpoints are included. Furthermore,it is to be understood that unless otherwise indicated or otherwiseevident from the context and understanding of one of ordinary skill inthe art, values that are expressed as ranges can assume any specificvalue or subrange within the stated ranges in different embodiments ofthe invention, to the tenth of the unit of the lower limit of the range,unless the context clearly dictates otherwise.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference. The citation of any publication is for its disclosure priorto the filing date and should not be construed as an admission that thepresent invention is not entitled to antedate such publication by virtueof prior invention.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that the various changes in form and detailsmay be made therein without departing from the scope of the inventionencompassed by the appended claims.

1. An oligonucleotide primer comprising a 3p arm having a 3′ end and a5′ end, a loop section and a 5p arm having a 3′ end and a 5′ end,wherein the 5p arm hybridizes to a DNA template and wherein the 3p armhybridizes to the DNA template and provides sequence specificity forpolymerase extension and wherein the loop section is located between the5p arm and the 3p arm and does not bind the DNA template.
 2. The primerof claim 1 wherein the DNA template is substantially complementary tothe 5p arm and the 3p arm.
 3. The primer of claim 1 wherein the 5p armis from 10 to 100 nucleotides in length.
 4. The primer of claim 1wherein the 3p arm is from 6 to 60 nucleotides in length.
 5. The primerof claim 1 wherein the loop section is from 12 to 50 nucleotides inlength.
 6. The primer of claim 1 wherein the 5p is from 25 to 60nucleotides in length and wherein the 3p arm is from 10 to 20nucleotides in length and wherein the loop section is from 15 to 40nucleotides in length.
 7. The primer of claim 1 wherein the 5′ end ofthe 3p arm and the 3′ end of the 5p arm are adjacent each other whenbound to the DNA template.
 8. The primer of claim 1 wherein the 5p armhas higher binding energy than the 3p arm when hybridized to the DNAtemplate.
 9. The primer of claim 1 wherein the 5p arm has a bindingenergy higher than twice that of the 3p arm.
 10. The primer of claim 1wherein the 5p arm has a binding energy higher than three times that ofthe 3p arm.
 11. The primer of claim 1 wherein the loop section is abulge loop.
 12. The primer of claim 1 wherein the loop section is ahairpin loop.
 13. The primer of claim 1 wherein the loop section is aninternal loop.
 14. A hybridization structure for assay use comprising aprobe and a target wherein the hybridization structure has one or moresingle stranded loops and two or more duplex segments wherein each loopis located between the duplex segments.
 15. The hybridization structureof claim 14 wherein the single stranded loop is in the probe andcomprises one or more non-nucleotide moieties.
 16. The hybridizationstructure of claim 14 wherein the probe comprises a spacer.
 17. Thehybridization structure of claim 14 wherein the hybridization structureis used for polymerase extension.
 18. The hybridization structure ofclaim 14 wherein the hybridization structure is used for hybridizationdetection.
 19. The hybridization structure of claim 18 wherein the loopcontains 12 to 50 nucleotides. 20-60. (canceled)